Cloning: Difference between revisions

From bradwiki
Jump to navigation Jump to search
 
(4 intermediate revisions by the same user not shown)
Line 3: Line 3:
Molecular cloning is similar to [https://en.wikipedia.org/wiki/Polymerase_chain_reaction polymerase chain reaction (PCR)] in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.
Molecular cloning is similar to [https://en.wikipedia.org/wiki/Polymerase_chain_reaction polymerase chain reaction (PCR)] in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.


==Plasmid Vector==
'''see also: [[Plasmid]]'''
A [https://en.wikipedia.org/wiki/Cloning_vector cloning vector] is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an [https://en.wikipedia.org/wiki/Expression_vector expression vector] is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.
A cloning vector almost always contains four DNA segments critically important to its function and experimental utility:
# DNA '''''replication origin''''' is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism
# one or more unique '''''restriction endonuclease recognition sites''''' to serve as sites where foreign DNA may be introduced
# a '''''selectable genetic marker''''' gene that can be used to enable the survival of cells that have taken up vector sequences
# a '''''tag''''' gene that can be used to screen for cells containing the foreign DNA


==Restriction Endonuclease==
==Restriction Endonuclease==
[[File:BamHI2.png|thumb|450px|Cleavage of a DNA sequence containing the BamHI restriction site. The DNA is cleaved at the palindromic sequence to produce 'sticky ends'.]]
A [https://en.wikipedia.org/wiki/Restriction_enzyme restriction enzyme] or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone strand of the DNA double helix.
A [https://en.wikipedia.org/wiki/Restriction_enzyme restriction enzyme] or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone strand of the DNA double helix.


Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and are a vital tool in [https://en.wikipedia.org/wiki/Molecular_cloning molecular cloning].
Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and are a vital tool in [https://en.wikipedia.org/wiki/Molecular_cloning molecular cloning].


==Plasmid Vector==
==Example Clone==
'''see also: [[Plasmid]]'''
 
{{ExpandBox|width=90%|float=left|opener=View|Recombinant DNA|
 
{{Style|font=courier|color=#000000|
ATTGACGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTACCATCACAATGGAGAAGCCAGTAGTAAACGTAGACGTAGACCCCCAGAGTCCGTTTG
TCGTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGCAGGTCACTCCAAATGACCATGCTAATGCCAGAGCATTTTCGCATCTGGCCAGTAAACTAA
TCGAGCTGGAGGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAATGTTTTCCGAGCACCAGTATCATTGTGTCTGCCCCATGCGTAGTC
CAGAAGACCCGGACCGCATGATGAAATACGCCAGTAAACTGGCGGAAAAAGCGTGCAAGATTACAAACAAGAACTTGCATGAGAAGATTAAGGATCTCCGGACCGTAC
TTGATACGCCGGATGCTGAAACACCATCGCTCTGCTTTCACAACGATGTTACCTGCAACATGCGTGCCGAATATTCCGTCATGCAGGACGTGTATATCAACGCTCCCG
GAACTATCTATCATCAGGCTATGAAAGGCGTGCGGACCCTGTACTGGATTGGCTTCGACACCACCCAGTTCATGTTCTCGGCTATGGCAGGTTCGTACCCTGCGTACA
ACACCAACTGGGCCGACGAGAAAGTCCTTGAAGCGCGTAACATCGGACTTTGCAGCACAAAGCTGAGTGAAGGTAGGACAGGAAAATTGTCGATAATGAGGAAGAAGG
AGTTGAAGCCCGGGTCGCGGGTTTATTTCTCCGTAGGATCGACACTTTATCCAGAACACAGAGCCAGCTTGCAGAGCTGGCATCTTCCATCGGTGTTCCACTTGAATG
GAAAGCAGTCGTACACTTGCCGCTGTGATACAGTGGTGAGTTGCGAAGGCTACGTAGTGAAGAAAATCACCATCAGTCCCGGGATCACGGGAGAAACCGTGGGATACG
CGGTTACACACAATAGCGAGGGCTTCTTGCTATGCAAAGTTACTGACACAGTAAAAGGAGAACGGGTATCGTTCCCTGTGTGCACGTACATCCCGGCCACCATATGCG
ATCAGATGACTGGTATAATGGCCACGGATATATCACCTGACGATGCACAAAAACTTCTGGTTGGGCTCAACCAGCGAATTGTCATTAACGGTAGGACTAACAGGAACA
CCAACACCATGCAAAATTACCTTCTGCCGATCATAGCACAAGGGTTCAGCAAATGGGCTAAGGAGCGCAAGGATGATCTTGATAACGAGAAAATGCTGGGTACTAGAG
AACGCAAGCTTACGTATGGCTGCTTGTGGGCGTTTCGCACTAAGAAAGTACATTCGTTTTATCGCCCACCTGGAACGCAGACCTGCGTAAAAGTCCCAGCCTCTTTTA
GCGCTTTTCCCATGTCGTCCGTATGGACGACCTCTTTGCCCATGTCGCTGAGGCAGAAATTGAAACTGGCATTGCAACCAAAGAAGGAGGAAAAACTGCTGCAGGTCT
CGGAGGAATTAGTCATGGAGGCCAAGGCTGCTTTTGAGGATGCTCAGGAGGAAGCCAGAGCGGAGAAGCTCCGAGAAGCACTTCCACCATTAGTGGCAGACAAAGGCA
TCGAGGCAGCCGCAGAAGTTGTCTGCGAAGTGGAGGGGCTCCAGGCGGACATCGGAGCAGCATTAGTTGAAACCCCGCGCGGTCACGTAAGGATAATACCTCAAGCAA
ATGACCGTATGATCGGACAGTATATCGTTGTCTCGCCAAACTCTGTGCTGAAGAATGCCAAACTCGCACCAGCGCACCCGCTAGCAGATCAGGTTAAGATCATAACAC
ACTCCGGAAGATCAGGAAGGTACGCGGTCGAACCATACGACGCTAAAGTACTGATGCCAGCAGGAGGTGCCGTACCATGGCCAGAATTCCTAGCACTGAGTGAGAGCG
CCACGTTAGTGTACAACGAAAGAGAGTTTGTGAACCGCAAACTATACCACATTGCCATGCATGGCCCCGCCAAGAATACAGAAGAGGAGCAGTACAAGGTTACAAAGG
CAGAGCTTGCAGAAACAGAGTACGTGTTTGACGTGGACAAGAAGCGTTGCGTTAAGAAGGAAGAAGCCTCAGGTCTGGTCCTCTCGGGAGAACTGACCAACCCTCCCT
ATCATGAGCTAGCTCTGGAGGGACTGAAGACCCGACCTGCGGTCCCGTACAAGGTCGAAACAATAGGAGTGATAGGCACACCGGGGTCGGGCAAGTCAGCTATTATCA
AGTCAACTGTCACGGCACGAGATCTTGTTACCAGCGGAAAGAAAGAAAATTGTCGCGAAATTGAGGCCGACGTGCTAAGACTGAGGGGTATGCAGATTACGTCGAAGA
CAGTAGATTCGGTTATGCTCAACGGATGCCACAAAGCCGTAGAAGTGCTGTACGTTGACGAAGCGTTCGCGTGCCACGCAGGAGCACTACTTGCCTTGATTGCTATCG
TCAGGCCCCGCAAGAAGGTAGTACTATGCGGAGACCCCATGCAATGCGGATTCTTCAACATGATGCAACTAAAGGTACATTTCAATCACCCTGAAAAAGACATATGCA
CCAAGACATTCTACAAGTATATCTCCCGGCGTTGCACACAGCCAGTTACAGCTATTGTATCGACACTGCATTACGATGGAAAGATGAAAACCACGAACCCGTGCAAGA
AGAACATTGAAATCGATATTACAGGGGCCACAAAGCCGAAGCCAGGGGATATCATCCTGACATGTTTCCGCGGGTGGGTTAAGCAATTGCAAATCGACTATCCCGGAC
ATGAAGTAATGACAGCCGCGGCCTCACAAGGGCTAACCAGAAAAGGAGTGTATGCCGTCCGGCAAAAAGTCAATGAAAACCCACTGTACGCGATCACATCAGAGCATG
TGAACGTGTTGCTCACCCGCACTGAGGACAGGCTAGTGTGGAAAACCTTGCAGGGCGACCCATGGATTAAGCAGCCCACTAACATACCTAAAGGAAACTTTCAGGCTA
CTATAGAGGACTGGGAAGCTGAACACAAGGGAATAATTGCTGCAATAAACAGCCCCACTCCCCGTGCCAATCCGTTCAGCTGCAAGACCAACGTTTGCTGGGCGAAAG
CATTGGAACCGATACTAGCCACGGCCGGTATCGTACTTACCGGTTGCCAGTGGAGCGAACTGTTCCCACAGTTTGCGGATGACAAACCACATTCGGCCATTTACGCCT
TAGACGTAATTTGCATTAAGTTTTTCGGCATGGACTTGACAAGCGGACTGTTTTCTAAACAGAGCATCCCACTAACGTACCATCCCGCCGATTCAGCGAGGCCGGTAG
CTCATTGGGACAACAGCCCAGGAACCCGCAAGTATGGGTACGATCACGCCATTGCCGCCGAACTCTCCCGTAGATTTCCGGTGTTCCAGCTAGCTGGGAAGGGCACAC
AACTTGATTTGCAGACGGGGAGAACCAGAGTTATCTCTGCACAGCATAACCTGGTCCCGGTGAACCGCAATCTTCCTCACGCCTTAGTCCCCGAGTACAAGGAGAAGC
AACCCGGCCCGGTCAAAAAATTCTTGAACCAGTTCAAACACCACTCAGTACTTGTGGTATCAGAGGAAAAAATTGAAGCTCCCCGTAAGAGAATCGAATGGATCGCCC
CGATTGGCATAGCCGGTGCAGATAAGAACTACAACCTGGCTTTCGGGTTTCCGCCGCAGGCACGGTACGACCTGGTGTTCATCAACATTGGAACTAAATACAGAAACC
ACCACTTTCAGCAGTGCGAAGACCATGCGGCGACCTTAAAAACCCTTTCGCGTTCGGCCCTGAATTGCCTTAACCCAGGAGGCACCCTCGTGGTGAAGTCCTATGGCT
ACGCCGACCGCAACAGTGAGGACGTAGTCACCGCTCTTGCCAGAAAGTTTGTCAGGGTGTCTGCAGCGAGACCAGATTGTGTCTCAAGCAATACAGAAATGTACCTGA
TTTTCCGACAACTAGACAACAGCCGTACACGGCAATTCACCCCGCACCATCTGAATTGCGTGATTTCGTCCGTGTATGAGGGTACAAGAGATGGAGTTGGAGCCGCGC
CGTCATACCGCACCAAAAGGGAGAATATTGCTGACTGTCAAGAGGAAGCAGTTGTCAACGCAGCCAATCCGCTGGGTAGACCAGGCGAAGGAGTCTGCCGTGCCATCT
ATAAACGTTGGCCGACCAGTTTTACCGATTCAGCCACGGAGACAGGCACCGCAAGAATGACTGTGTGCCTAGGAAAGAAAGTGATCCACGCGGTCGGCCCTGATTTCC
GGAAGCACCCAGAAGCAGAAGCCTTGAAATTGCTACAAAACGCCTACCATGCAGTGGCAGACTTAGTAAATGAACATAACATCAAGTCTGTCGCCATTCCACTGCTAT
CTACAGGCATTTACGCAGCCGGAAAAGACCGCCTTGAAGTATCACTTAACTGCTTGACAACCGCGCTAGACAGAACTGACGCGGACGTAACCATCTATTGCCTGGATA
AGAAGTGGAAGGAAAGAATCGACGCGGCACTCCAACTTAAGGAGTCTGTAACAGAGCTGAAGGATGAAGATATGGAGATCGACGATGAGTTAGTATGGATTCATCCAG
ACAGTTGCTTGAAGGGAAGAAAGGGATTCAGTACTACAAAAGGAAAATTGTATTCGTACTTCGAAGGCACCAAATTCCATCAAGCAGCAAAAGACATGGCGGAGATAA
AGGTCCTGTTCCCTAATGACCAGGAAAGTAATGAACAACTGTGTGCCTACATATTGGGTGAGACCATGGAAGCAATCCGCGAAAAGTGCCCGGTCGACCATAACCCGT
CGTCTAGCCCGCCCAAAACGTTGCCGTGCCTTTGCATGTATGCCATGACGCCAGAAAGGGTCCACAGACTTAGAAGCAATAACGTCAAAGAAGTTACAGTATGCTCCT
CCACCCCCCTTCCTAAGCACAAAATTAAGAATGTTCAGAAGGTTCAGTGCACGAAAGTAGTCCTGTTTAATCCGCACACTCCCGCATTCGTTCCCGCCCGTAAGTACA
TAGAAGTGCCAGAACAGCCTACCGCTCCTCCTGCACAGGCCGAGGAGGCCCCCGAAGTTGTAGCGACACCGTCACCATCTACAGCTGATAACACCTCGCTTGATGTCA
CAGACATCTCACTGGATATGGATGACAGTAGCGAAGGCTCACTTTTTTCGAGCTTTAGCGGATCGGACAACTCTATTACTAGTATGGACAGTTGGTCGTCAGGACCTA
GTTCACTAGAGATAGTAGACCGAAGGCAGGTGGTGGTGGCTGACGTTCATGCCGTCCAAGAGCCTGCCCCTATTCCACCGCCAAGGCTAAAGAAGATGGCCCGCCTGG
CAGCGGCAAGAAAAGAGCCCACTCCACCGGCAAGCAATAGCTCTGAGTCCCTCCACCTCTCTTTTGGTGGGGTATCCATGTCCCTCGGATCAATTTTCGACGGAGAGA
CGGCCCGCCAGGCAGCGGTACAACCCCTGGCAACAGGCCCCACGGATGTGCCTATGTCTTTCGGATCGTTTTCCGACGGAGAGATTGATGAGCTGAGCCGCAGAGTAA
CTGAGTCCGAACCCGTCCTGTTTGGATCATTTGAACCGGGCGAAGTGAACTCAATTATATCGTCCCGATCAGCCGTATCTTTTCCACTACGCAAGCAGAGACGTAGAC
GCAGGAGCAGGAGGACTGAATACTGACTAACCGGGGTAGGTGGGTACATATTTTCGACGGACACAGGCCCTGGGCACTTGCAAAAGAAGTCCGTTCTGCAGAACCAGC
TTACAGAACCGACCTTGGAGCGCAATGTCCTGGAAAGAATTCATGCCCCGGTGCTCGACACGTCGAAAGAGGAACAACTCAAACTCAGGTACCAGATGATGCCCACCG
AAGCCAACAAAAGTAGGTACCAGTCTCGTAAAGTAGAAAATCAGAAAGCCATAACCACTGAGCGACTACTGTCAGGACTACGACTGTATAACTCTGCCACAGATCAGC
CAGAATGCTATAAGATCACCTATCCGAAACCATTGTACTCCAGTAGCGTACCGGCGAACTACTCCGATCCACAGTTCGCTGTAGCTGTCTGTAACAACTATCTGCATG
AGAACTATCCGACAGTAGCATCTTATCAGATTACTGACGAGTACGATGCTTACTTGGATATGGTAGACGGGACAGTCGCCTGCCTGGATACTGCAACCTTCTGCCCCG
CTAAGCTTAGAAGTTACCCGAAAAAACATGAGTATAGAGCCCCGAATATCCGCAGTGCGGTTCCATCAGCGATGCAGAACACGCTACAAAATGTGCTCATTGCCGCAA
CTAAAAGAAATTGCAACGTCACGCAGATGCGTGAACTGCCAACACTGGACTCAGCGACATTCAATGTCGAATGCTTTCGAAAATATGCATGTAATGACGAGTATTGGG
AGGAGTTCGCTCGGAAGCCAATTAGGATTACCACTGAGTTTGTCACCGCATATGTAGCTAGACTGAAAGGCCCTAAGGCCGCCGCACTATTTGCAAAGACGTATAATT
TGGTCCCATTGCAAGAAGTGCCTATGGATAGATTCGTCATGGACATGAAAAGAGACGTGAAAGTTACACCAGGCACGAAACACACAGAAGAAAGACCGAAAGTACAAG
TGATACAAGCCGCAGAACCCCTGGCGACTGCTTACTTATGCGGGATTCACCGGGAATTAGTGCGTAGGCTTACGGCCGTCTTGCTTCCAAACATTCACACGCTTTTTG
ACATGTCGGCGGAGGATTTTGATGCAATCATAGCAGAACACTTCAAGCAAGGCGACCCGGTACTGGAGACGGATATCGCATCATTCGACAAAAGCCAAGACGACGCTA
TGGCGTTAACCGGTCTGATGATCTTGGAGGACCTGGGTGTGGATCAACCACTACTCGACTTGATCGAGTGCGCCTTTGGAGAAATATCATCCACCCATCTACCTACGG
GTACTCGTTTTAAATTCGGGGCGATGATGAAATCCGGAATGTTCCTCACACTTTTTGTCAACACAGTTTTGAATGTCGTTATCGCCAGCAGAGTACTAGAAGAGCGGC
TTAAAACGTCCAGATGTGCAGCGTTCATTGGCGACGACAACATCATACATGGAGTAGTATCTGACAAAGAAATGGCTGAGAGGTGCGCCACCTGGCTCAACATGGAGG
TTAAGATCATCGACGCAGTCATCGGTGAGAGACCACCTTACTTCTGCGGCGGATTTATCTTGCAAGATTCGGTTACTTCCACAGCGTGCCGCGTGGCGGATCCCCTGA
AAAGGCTGTTTAAGTTGGGTAAACCGCTCCCAGCCGACGACGAGCAAGACGAAGACAGAAGACGCGCTCTGCTAGATGAAACAAAGGCGTGGTTTAGAGTAGGTATAA
CAGGCACTTTAGCAGTGGCCGTGACGACCCGGTATGAGGTAGACAATATTACACCTGTCCTACTGGCATTGAGAACTTTTGCCCAGAGCAAAAGAGCATTCCAAGCCA
TCAGAGGGGAAATTAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAGACCACCATGGTGAGCA
AGGGCGAGGAGAATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGG
GCCGCCCCTACGAGGCCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGG
TCTACATTAAGCACCCAGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAGGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCATTATTCACG
TTAACCAGGACTCCTCCCTGCAGGACGGCGTGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCT
GGGAGGCCTCCGAGGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGAGCGAGATCAAGAAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGCCGCCGAGGTCAAGA
CCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACATCGTCGACATCAAGTTGGACATCGTGTCCCACAACGAGGACTACACCATCGTGGAACAGTACG
AACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCCATGGCCGGGTGGAACGCCTACATCGACA
GCCTTATGGCGGACGGGACCTGTCAGGACGCGGCCATCGTAGGCTACAAGGACTCGCCCTCCGTCTGGGCCGCCGTCCCCGGGAAGACCTTCGTTAGCATTACGCCAG
CTGAGGTTGGTGTCCTGGTAGGCAAAGACCGGTCAAGTTTTTTCGTCAATGGGCTGACACTTGGGGGCCAGAAATGTTCTGTGATCCGGGACTCACTGCTGCAAGACG
GGGAATTTACAATGGATCTTCGTACCAAGAGCACCGGAGGAGCCCCCACCTTCAATGTCACTGTCACCATGACTGCCAAGACGCTAGTCCTGCTGATGGGCAAAGAAG
GTGTCCACGGTGGTTTGATCAACAAGAAATGTTATGAAATGGCCTCTCACCTGCGGCGTTCCCAGTACTAAACCGCATGCAGGCCTTAAAGCATCTCTACGGTGGTCC
TAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAGTAGAGGCGCACCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTG
CCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC
ACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTC
AAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATCTTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTG
AATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAG
GCCAAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCT
GTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTACTTCGGCTTCGTGACCGCCGCCGCC
ATCACCCACGGCATGGATGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCC
GGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGC
GACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTAC
AATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTC
AACACCCCAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGCATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTG
CCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACTGACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGC
TTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTCC
CTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCC
TGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCT
GGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGC
TCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCTTCTAGGGGCCCAAT
GATCCGACCAGCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAACACTAAAAA
CTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGTTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATTTAGCGGACGCCAAAAACTCAATGTATTTTTG
AGGAAGCGTGGTGCATAATGCCACGCAGGTTTTGCATAATTTTTTTTTTTTTTTTTATTAATCAACAAAATTTTGTTTTTAACATTTCAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAGGGGAATTCCTCGATAATTAAGCGGCCGCTTAATTAATCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATG
TGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTA
TGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC
AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC
TGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAA
AGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGG
AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC
CTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC
CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC
CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT
CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC
CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT
AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA
CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA
GATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC
TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA
ACGCCAGCAACGCGAGCTCGTATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGGGACACTATAG
}}
 
{{Style|font=courier|color=#FF0080|TCTAGA}}
 
{{Style|font=courier|color=#000000|CCACC}}
 
{{Style|font=courier|color=#003D99|
ATGGTGAGCAAGGGCGAGGAGAATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGC
GAGGGCGAGGGCCGCCCCTACGAGGCCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTAC
GGCTCCAAGGTCTACATTAAGCACCCAGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAGGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC
ATTATTCACGTTAACCAGGACTCCTCCCTGCAGGACGGCGTGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAG
ACCATGGGCTGGGAGGCCTCCGAGGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGAGCGAGATCAAGAAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGCCGCC
GAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACATCGTCGACATCAAGTTGGACATCGTGTCCCACAACGAGGACTACACCATCGTG
GAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCCATGGCCGGGTGGAACGCC
TACATCGACAGCCTTATGGCGGACGGGACCTGTCAGGACGCGGCCATCGTAGGCTACAAGGACTCGCCCTCCGTCTGGGCCGCCGTCCCCGGGAAGACCTTCGTTAGC
ATTACGCCAGCTGAGGTTGGTGTCCTGGTAGGCAAAGACCGGTCAAGTTTTTTCGTCAATGGGCTGACACTTGGGGGCCAGAAATGTTCTGTGATCCGGGACTCACTG
CTGCAAGACGGGGAATTTACAATGGATCTTCGTACCAAGAGCACCGGAGGAGCCCCCACCTTCAATGTCACTGTCACCATGACTGCCAAGACGCTAGTCCTGCTGATG
GGCAAAGAAGGTGTCCACGGTGGTTTGATCAACAAGAAATGTTATGAAATGGCCTCTCACCTGCGGCGTTCCCAGTACTAA
}}
 
{{Style|font=courier|color=#000000|ACC}}
 
{{Style|font=courier|color=#FF0080|GCATGC}}


A [https://en.wikipedia.org/wiki/Cloning_vector cloning vector] is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
{{Style|font=courier|color=#000000|AGGCCT}}


{{Style|font=courier|color=#B24700|
TAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAG
}}


Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an [https://en.wikipedia.org/wiki/Expression_vector expression vector] is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.
{{Style|font=courier|color=#000000|TAGAGGCGCACC}}


==Example Clone==
{{Style|font=courier|color=#003D99|
ATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGC
GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTC
TCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATCTTCTTCGAGGATGACGGCAACTACAAG
TCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTGAATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTAC
AACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCCAAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCC
GACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCTGTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGC
GATCACATGATCTACTTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTATGGATGATGATATCGCC
GCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGC
GTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAAC
TGGGACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCC
AACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACACCCCAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGC
ATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTGCCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACT
GACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGAC
TTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTCCCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGCCCT
GAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTAC
GCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATT
GCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGC
CCCTCCATCGTCCACCGCAAATGCTTCTAG
}}


ATTGACGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTACCATCACAATGGAGAAGCCAGTAGTAAACGTAGACGTAGACCC
CCAGAGTCCGTTTGTCGTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGCAGGTCACTCCAAATGACCATGCTAATGCCAGA
GCATTTTCGCATCTGGCCAGTAAACTAATCGAGCTGGAGGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAATGT
TTTCCGAGCACCAGTATCATTGTGTCTGCCCCATGCGTAGTCCAGAAGACCCGGACCGCATGATGAAATACGCCAGTAAACTGGCGGAAAAAGC
GTGCAAGATTACAAACAAGAACTTGCATGAGAAGATTAAGGATCTCCGGACCGTACTTGATACGCCGGATGCTGAAACACCATCGCTCTGCTTT
CACAACGATGTTACCTGCAACATGCGTGCCGAATATTCCGTCATGCAGGACGTGTATATCAACGCTCCCGGAACTATCTATCATCAGGCTATGA
AAGGCGTGCGGACCCTGTACTGGATTGGCTTCGACACCACCCAGTTCATGTTCTCGGCTATGGCAGGTTCGTACCCTGCGTACAACACCAACTG
GGCCGACGAGAAAGTCCTTGAAGCGCGTAACATCGGACTTTGCAGCACAAAGCTGAGTGAAGGTAGGACAGGAAAATTGTCGATAATGAGGAAG
AAGGAGTTGAAGCCCGGGTCGCGGGTTTATTTCTCCGTAGGATCGACACTTTATCCAGAACACAGAGCCAGCTTGCAGAGCTGGCATCTTCCAT
CGGTGTTCCACTTGAATGGAAAGCAGTCGTACACTTGCCGCTGTGATACAGTGGTGAGTTGCGAAGGCTACGTAGTGAAGAAAATCACCATCAG
TCCCGGGATCACGGGAGAAACCGTGGGATACGCGGTTACACACAATAGCGAGGGCTTCTTGCTATGCAAAGTTACTGACACAGTAAAAGGAGAA
CGGGTATCGTTCCCTGTGTGCACGTACATCCCGGCCACCATATGCGATCAGATGACTGGTATAATGGCCACGGATATATCACCTGACGATGCAC
AAAAACTTCTGGTTGGGCTCAACCAGCGAATTGTCATTAACGGTAGGACTAACAGGAACACCAACACCATGCAAAATTACCTTCTGCCGATCAT
AGCACAAGGGTTCAGCAAATGGGCTAAGGAGCGCAAGGATGATCTTGATAACGAGAAAATGCTGGGTACTAGAGAACGCAAGCTTACGTATGGC
TGCTTGTGGGCGTTTCGCACTAAGAAAGTACATTCGTTTTATCGCCCACCTGGAACGCAGACCTGCGTAAAAGTCCCAGCCTCTTTTAGCGCTT
TTCCCATGTCGTCCGTATGGACGACCTCTTTGCCCATGTCGCTGAGGCAGAAATTGAAACTGGCATTGCAACCAAAGAAGGAGGAAAAACTGCT
GCAGGTCTCGGAGGAATTAGTCATGGAGGCCAAGGCTGCTTTTGAGGATGCTCAGGAGGAAGCCAGAGCGGAGAAGCTCCGAGAAGCACTTCCA
CCATTAGTGGCAGACAAAGGCATCGAGGCAGCCGCAGAAGTTGTCTGCGAAGTGGAGGGGCTCCAGGCGGACATCGGAGCAGCATTAGTTGAAA
CCCCGCGCGGTCACGTAAGGATAATACCTCAAGCAAATGACCGTATGATCGGACAGTATATCGTTGTCTCGCCAAACTCTGTGCTGAAGAATGC
CAAACTCGCACCAGCGCACCCGCTAGCAGATCAGGTTAAGATCATAACACACTCCGGAAGATCAGGAAGGTACGCGGTCGAACCATACGACGCT
AAAGTACTGATGCCAGCAGGAGGTGCCGTACCATGGCCAGAATTCCTAGCACTGAGTGAGAGCGCCACGTTAGTGTACAACGAAAGAGAGTTTG
TGAACCGCAAACTATACCACATTGCCATGCATGGCCCCGCCAAGAATACAGAAGAGGAGCAGTACAAGGTTACAAAGGCAGAGCTTGCAGAAAC
AGAGTACGTGTTTGACGTGGACAAGAAGCGTTGCGTTAAGAAGGAAGAAGCCTCAGGTCTGGTCCTCTCGGGAGAACTGACCAACCCTCCCTAT
CATGAGCTAGCTCTGGAGGGACTGAAGACCCGACCTGCGGTCCCGTACAAGGTCGAAACAATAGGAGTGATAGGCACACCGGGGTCGGGCAAGT
CAGCTATTATCAAGTCAACTGTCACGGCACGAGATCTTGTTACCAGCGGAAAGAAAGAAAATTGTCGCGAAATTGAGGCCGACGTGCTAAGACT
GAGGGGTATGCAGATTACGTCGAAGACAGTAGATTCGGTTATGCTCAACGGATGCCACAAAGCCGTAGAAGTGCTGTACGTTGACGAAGCGTTC
GCGTGCCACGCAGGAGCACTACTTGCCTTGATTGCTATCGTCAGGCCCCGCAAGAAGGTAGTACTATGCGGAGACCCCATGCAATGCGGATTCT
TCAACATGATGCAACTAAAGGTACATTTCAATCACCCTGAAAAAGACATATGCACCAAGACATTCTACAAGTATATCTCCCGGCGTTGCACACA
GCCAGTTACAGCTATTGTATCGACACTGCATTACGATGGAAAGATGAAAACCACGAACCCGTGCAAGAAGAACATTGAAATCGATATTACAGGG
GCCACAAAGCCGAAGCCAGGGGATATCATCCTGACATGTTTCCGCGGGTGGGTTAAGCAATTGCAAATCGACTATCCCGGACATGAAGTAATGA
CAGCCGCGGCCTCACAAGGGCTAACCAGAAAAGGAGTGTATGCCGTCCGGCAAAAAGTCAATGAAAACCCACTGTACGCGATCACATCAGAGCA
TGTGAACGTGTTGCTCACCCGCACTGAGGACAGGCTAGTGTGGAAAACCTTGCAGGGCGACCCATGGATTAAGCAGCCCACTAACATACCTAAA
GGAAACTTTCAGGCTACTATAGAGGACTGGGAAGCTGAACACAAGGGAATAATTGCTGCAATAAACAGCCCCACTCCCCGTGCCAATCCGTTCA
GCTGCAAGACCAACGTTTGCTGGGCGAAAGCATTGGAACCGATACTAGCCACGGCCGGTATCGTACTTACCGGTTGCCAGTGGAGCGAACTGTT
CCCACAGTTTGCGGATGACAAACCACATTCGGCCATTTACGCCTTAGACGTAATTTGCATTAAGTTTTTCGGCATGGACTTGACAAGCGGACTG
TTTTCTAAACAGAGCATCCCACTAACGTACCATCCCGCCGATTCAGCGAGGCCGGTAGCTCATTGGGACAACAGCCCAGGAACCCGCAAGTATG
GGTACGATCACGCCATTGCCGCCGAACTCTCCCGTAGATTTCCGGTGTTCCAGCTAGCTGGGAAGGGCACACAACTTGATTTGCAGACGGGGAG
AACCAGAGTTATCTCTGCACAGCATAACCTGGTCCCGGTGAACCGCAATCTTCCTCACGCCTTAGTCCCCGAGTACAAGGAGAAGCAACCCGGC
CCGGTCAAAAAATTCTTGAACCAGTTCAAACACCACTCAGTACTTGTGGTATCAGAGGAAAAAATTGAAGCTCCCCGTAAGAGAATCGAATGGA
TCGCCCCGATTGGCATAGCCGGTGCAGATAAGAACTACAACCTGGCTTTCGGGTTTCCGCCGCAGGCACGGTACGACCTGGTGTTCATCAACAT
TGGAACTAAATACAGAAACCACCACTTTCAGCAGTGCGAAGACCATGCGGCGACCTTAAAAACCCTTTCGCGTTCGGCCCTGAATTGCCTTAAC
CCAGGAGGCACCCTCGTGGTGAAGTCCTATGGCTACGCCGACCGCAACAGTGAGGACGTAGTCACCGCTCTTGCCAGAAAGTTTGTCAGGGTGT
CTGCAGCGAGACCAGATTGTGTCTCAAGCAATACAGAAATGTACCTGATTTTCCGACAACTAGACAACAGCCGTACACGGCAATTCACCCCGCA
CCATCTGAATTGCGTGATTTCGTCCGTGTATGAGGGTACAAGAGATGGAGTTGGAGCCGCGCCGTCATACCGCACCAAAAGGGAGAATATTGCT
GACTGTCAAGAGGAAGCAGTTGTCAACGCAGCCAATCCGCTGGGTAGACCAGGCGAAGGAGTCTGCCGTGCCATCTATAAACGTTGGCCGACCA
GTTTTACCGATTCAGCCACGGAGACAGGCACCGCAAGAATGACTGTGTGCCTAGGAAAGAAAGTGATCCACGCGGTCGGCCCTGATTTCCGGAA
GCACCCAGAAGCAGAAGCCTTGAAATTGCTACAAAACGCCTACCATGCAGTGGCAGACTTAGTAAATGAACATAACATCAAGTCTGTCGCCATT
CCACTGCTATCTACAGGCATTTACGCAGCCGGAAAAGACCGCCTTGAAGTATCACTTAACTGCTTGACAACCGCGCTAGACAGAACTGACGCGG
ACGTAACCATCTATTGCCTGGATAAGAAGTGGAAGGAAAGAATCGACGCGGCACTCCAACTTAAGGAGTCTGTAACAGAGCTGAAGGATGAAGA
TATGGAGATCGACGATGAGTTAGTATGGATTCATCCAGACAGTTGCTTGAAGGGAAGAAAGGGATTCAGTACTACAAAAGGAAAATTGTATTCG
TACTTCGAAGGCACCAAATTCCATCAAGCAGCAAAAGACATGGCGGAGATAAAGGTCCTGTTCCCTAATGACCAGGAAAGTAATGAACAACTGT
GTGCCTACATATTGGGTGAGACCATGGAAGCAATCCGCGAAAAGTGCCCGGTCGACCATAACCCGTCGTCTAGCCCGCCCAAAACGTTGCCGTG
CCTTTGCATGTATGCCATGACGCCAGAAAGGGTCCACAGACTTAGAAGCAATAACGTCAAAGAAGTTACAGTATGCTCCTCCACCCCCCTTCCT
AAGCACAAAATTAAGAATGTTCAGAAGGTTCAGTGCACGAAAGTAGTCCTGTTTAATCCGCACACTCCCGCATTCGTTCCCGCCCGTAAGTACA
TAGAAGTGCCAGAACAGCCTACCGCTCCTCCTGCACAGGCCGAGGAGGCCCCCGAAGTTGTAGCGACACCGTCACCATCTACAGCTGATAACAC
CTCGCTTGATGTCACAGACATCTCACTGGATATGGATGACAGTAGCGAAGGCTCACTTTTTTCGAGCTTTAGCGGATCGGACAACTCTATTACT
AGTATGGACAGTTGGTCGTCAGGACCTAGTTCACTAGAGATAGTAGACCGAAGGCAGGTGGTGGTGGCTGACGTTCATGCCGTCCAAGAGCCTG
CCCCTATTCCACCGCCAAGGCTAAAGAAGATGGCCCGCCTGGCAGCGGCAAGAAAAGAGCCCACTCCACCGGCAAGCAATAGCTCTGAGTCCCT
CCACCTCTCTTTTGGTGGGGTATCCATGTCCCTCGGATCAATTTTCGACGGAGAGACGGCCCGCCAGGCAGCGGTACAACCCCTGGCAACAGGC
CCCACGGATGTGCCTATGTCTTTCGGATCGTTTTCCGACGGAGAGATTGATGAGCTGAGCCGCAGAGTAACTGAGTCCGAACCCGTCCTGTTTG
GATCATTTGAACCGGGCGAAGTGAACTCAATTATATCGTCCCGATCAGCCGTATCTTTTCCACTACGCAAGCAGAGACGTAGACGCAGGAGCAG
GAGGACTGAATACTGACTAACCGGGGTAGGTGGGTACATATTTTCGACGGACACAGGCCCTGGGCACTTGCAAAAGAAGTCCGTTCTGCAGAAC
CAGCTTACAGAACCGACCTTGGAGCGCAATGTCCTGGAAAGAATTCATGCCCCGGTGCTCGACACGTCGAAAGAGGAACAACTCAAACTCAGGT
ACCAGATGATGCCCACCGAAGCCAACAAAAGTAGGTACCAGTCTCGTAAAGTAGAAAATCAGAAAGCCATAACCACTGAGCGACTACTGTCAGG
ACTACGACTGTATAACTCTGCCACAGATCAGCCAGAATGCTATAAGATCACCTATCCGAAACCATTGTACTCCAGTAGCGTACCGGCGAACTAC
TCCGATCCACAGTTCGCTGTAGCTGTCTGTAACAACTATCTGCATGAGAACTATCCGACAGTAGCATCTTATCAGATTACTGACGAGTACGATG
CTTACTTGGATATGGTAGACGGGACAGTCGCCTGCCTGGATACTGCAACCTTCTGCCCCGCTAAGCTTAGAAGTTACCCGAAAAAACATGAGTA
TAGAGCCCCGAATATCCGCAGTGCGGTTCCATCAGCGATGCAGAACACGCTACAAAATGTGCTCATTGCCGCAACTAAAAGAAATTGCAACGTC
ACGCAGATGCGTGAACTGCCAACACTGGACTCAGCGACATTCAATGTCGAATGCTTTCGAAAATATGCATGTAATGACGAGTATTGGGAGGAGT
TCGCTCGGAAGCCAATTAGGATTACCACTGAGTTTGTCACCGCATATGTAGCTAGACTGAAAGGCCCTAAGGCCGCCGCACTATTTGCAAAGAC
GTATAATTTGGTCCCATTGCAAGAAGTGCCTATGGATAGATTCGTCATGGACATGAAAAGAGACGTGAAAGTTACACCAGGCACGAAACACACA
GAAGAAAGACCGAAAGTACAAGTGATACAAGCCGCAGAACCCCTGGCGACTGCTTACTTATGCGGGATTCACCGGGAATTAGTGCGTAGGCTTA
CGGCCGTCTTGCTTCCAAACATTCACACGCTTTTTGACATGTCGGCGGAGGATTTTGATGCAATCATAGCAGAACACTTCAAGCAAGGCGACCC
GGTACTGGAGACGGATATCGCATCATTCGACAAAAGCCAAGACGACGCTATGGCGTTAACCGGTCTGATGATCTTGGAGGACCTGGGTGTGGAT
CAACCACTACTCGACTTGATCGAGTGCGCCTTTGGAGAAATATCATCCACCCATCTACCTACGGGTACTCGTTTTAAATTCGGGGCGATGATGA
AATCCGGAATGTTCCTCACACTTTTTGTCAACACAGTTTTGAATGTCGTTATCGCCAGCAGAGTACTAGAAGAGCGGCTTAAAACGTCCAGATG
TGCAGCGTTCATTGGCGACGACAACATCATACATGGAGTAGTATCTGACAAAGAAATGGCTGAGAGGTGCGCCACCTGGCTCAACATGGAGGTT
AAGATCATCGACGCAGTCATCGGTGAGAGACCACCTTACTTCTGCGGCGGATTTATCTTGCAAGATTCGGTTACTTCCACAGCGTGCCGCGTGG
CGGATCCCCTGAAAAGGCTGTTTAAGTTGGGTAAACCGCTCCCAGCCGACGACGAGCAAGACGAAGACAGAAGACGCGCTCTGCTAGATGAAAC
AAAGGCGTGGTTTAGAGTAGGTATAACAGGCACTTTAGCAGTGGCCGTGACGACCCGGTATGAGGTAGACAATATTACACCTGTCCTACTGGCA
TTGAGAACTTTTGCCCAGAGCAAAAGAGCATTCCAAGCCATCAGAGGGGAAATTAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTAC
ATTTCATCTGACTAATACCACAACACCACCACCTCTAGACCACCATGGTGAGCAAGGGCGAGGAGAATAACATGGCCATCATCAAGGAGTTCAT
GCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGCCTTTCAGACC
GCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGTCTACATTAAGC
ACCCAGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAGGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCATTATTCA
CGTTAACCAGGACTCCTCCCTGCAGGACGGCGTGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAG
AAGAAGACCATGGGCTGGGAGGCCTCCGAGGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGAGCGAGATCAAGAAGAGGCTGAAGCTGAAGG
ACGGCGGCCACTACGCCGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACATCGTCGACATCAAGTTGGA
CATCGTGTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG
TCCGGACTCAGATCTCGAGCTCAAGCTTCCATGGCCGGGTGGAACGCCTACATCGACAGCCTTATGGCGGACGGGACCTGTCAGGACGCGGCCA
TCGTAGGCTACAAGGACTCGCCCTCCGTCTGGGCCGCCGTCCCCGGGAAGACCTTCGTTAGCATTACGCCAGCTGAGGTTGGTGTCCTGGTAGG
CAAAGACCGGTCAAGTTTTTTCGTCAATGGGCTGACACTTGGGGGCCAGAAATGTTCTGTGATCCGGGACTCACTGCTGCAAGACGGGGAATTT
ACAATGGATCTTCGTACCAAGAGCACCGGAGGAGCCCCCACCTTCAATGTCACTGTCACCATGACTGCCAAGACGCTAGTCCTGCTGATGGGCA
AAGAAGGTGTCCACGGTGGTTTGATCAACAAGAAATGTTATGAAATGGCCTCTCACCTGCGGCGTTCCCAGTACTAAACCGCATGCAGGCCTTA
AAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAGTAGAGGCGCACCATGGT
GAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAG
GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCC
TGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGA
GCGCACCATCTTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTGAATCGCATCGAGCTGACC
GGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCCA
AGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAATACCCCCATCGG
CGATGGCCCTGTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTAC
TTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTATGGATGATGATATCGCCG
CGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCC
CAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTAC
CCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGC
ACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACACCCCAGCCAT
GTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGCATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTG
CCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACTGACTACCTCATGAAGATCCTCACCG
AGCGCGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAAGA
GATGGCCACGGCTGCTTCCAGCTCCTCCCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGC
CCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACA
TCCGCAAAGACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCT
GGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACC
TTCCAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCTTCTAGGGGCCCAATGATCCGACCA
GCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAACACTA
AAAACTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGTTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATTTAGCGGACGCCAA
AAACTCAATGTATTTTTGAGGAAGCGTGGTGCATAATGCCACGCAGGTTTTGCATAATTTTTTTTTTTTTTTTTATTAATCAACAAAATTTTGT
TTTTAACATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGAATTCCTCGATAATTAAGCGGCCGCTTAATTAATCGAGGGGAAT
TAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT
ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCC
CTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT
TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT
GTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT
CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG
ACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATG
AAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC
TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGAT
AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG
GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTC
ATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA
CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC
AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA
GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTG
TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA
CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCC
CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT
TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG
CGAGCTCGTATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGGGACACTATAG


{{Style|font=courier|color=#000000|
GGGCCCAATGATCCGACCAGCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAA
CACTAAAAACTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGTTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATTTAGCGGACGCCAAAAACTCAAT
GTATTTTTGAGGAAGCGTGGTGCATAATGCCACGCAGGTTTTGCATAATTTTTTTTTTTTTTTTTATTAATCAACAAAATTTTGTTTTTAACATTTC
}}




{{Style|font=courier|color=#248F24|
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
}}




{{Style|font=courier|color=#000000|
GGGGAATTCCTCGATAATTAAGCGGCCGCTTAATTAATCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCT
ATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT
TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA
GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCG
GTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT
GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTT
TTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCA
ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG
GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA
GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC
TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG
TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG
GTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC
CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA
AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT
GAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAAC
GCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG
AGCTCGTATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGGGACACTATAG
}}


}}




[[Category:Neurobiology]] [[Category:Synaptic Plasticity]] [[Category:Neuroinformatics]] [[Category:Genetics]] [[Category:Neuroscience Methods]] [[Category:Methods]]
[[Category:Neurobiology]] [[Category:Synaptic Plasticity]] [[Category:Neuroinformatics]] [[Category:Genetics]] [[Category:Neuroscience Methods]] [[Category:Methods]]

Latest revision as of 02:24, 11 December 2015

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.


Plasmid Vector

see also: Plasmid

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.


Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.


A cloning vector almost always contains four DNA segments critically important to its function and experimental utility:

  1. DNA replication origin is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism
  2. one or more unique restriction endonuclease recognition sites to serve as sites where foreign DNA may be introduced
  3. a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences
  4. a tag gene that can be used to screen for cells containing the foreign DNA

Restriction Endonuclease

Error creating thumbnail: File missing
Cleavage of a DNA sequence containing the BamHI restriction site. The DNA is cleaved at the palindromic sequence to produce 'sticky ends'.

A restriction enzyme or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone strand of the DNA double helix.

Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and are a vital tool in molecular cloning.

Example Clone

Recombinant DNA



ATTGACGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTACCATCACAATGGAGAAGCCAGTAGTAAACGTAGACGTAGACCCCCAGAGTCCGTTTG TCGTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGCAGGTCACTCCAAATGACCATGCTAATGCCAGAGCATTTTCGCATCTGGCCAGTAAACTAA TCGAGCTGGAGGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAATGTTTTCCGAGCACCAGTATCATTGTGTCTGCCCCATGCGTAGTC CAGAAGACCCGGACCGCATGATGAAATACGCCAGTAAACTGGCGGAAAAAGCGTGCAAGATTACAAACAAGAACTTGCATGAGAAGATTAAGGATCTCCGGACCGTAC TTGATACGCCGGATGCTGAAACACCATCGCTCTGCTTTCACAACGATGTTACCTGCAACATGCGTGCCGAATATTCCGTCATGCAGGACGTGTATATCAACGCTCCCG GAACTATCTATCATCAGGCTATGAAAGGCGTGCGGACCCTGTACTGGATTGGCTTCGACACCACCCAGTTCATGTTCTCGGCTATGGCAGGTTCGTACCCTGCGTACA ACACCAACTGGGCCGACGAGAAAGTCCTTGAAGCGCGTAACATCGGACTTTGCAGCACAAAGCTGAGTGAAGGTAGGACAGGAAAATTGTCGATAATGAGGAAGAAGG AGTTGAAGCCCGGGTCGCGGGTTTATTTCTCCGTAGGATCGACACTTTATCCAGAACACAGAGCCAGCTTGCAGAGCTGGCATCTTCCATCGGTGTTCCACTTGAATG GAAAGCAGTCGTACACTTGCCGCTGTGATACAGTGGTGAGTTGCGAAGGCTACGTAGTGAAGAAAATCACCATCAGTCCCGGGATCACGGGAGAAACCGTGGGATACG CGGTTACACACAATAGCGAGGGCTTCTTGCTATGCAAAGTTACTGACACAGTAAAAGGAGAACGGGTATCGTTCCCTGTGTGCACGTACATCCCGGCCACCATATGCG ATCAGATGACTGGTATAATGGCCACGGATATATCACCTGACGATGCACAAAAACTTCTGGTTGGGCTCAACCAGCGAATTGTCATTAACGGTAGGACTAACAGGAACA CCAACACCATGCAAAATTACCTTCTGCCGATCATAGCACAAGGGTTCAGCAAATGGGCTAAGGAGCGCAAGGATGATCTTGATAACGAGAAAATGCTGGGTACTAGAG AACGCAAGCTTACGTATGGCTGCTTGTGGGCGTTTCGCACTAAGAAAGTACATTCGTTTTATCGCCCACCTGGAACGCAGACCTGCGTAAAAGTCCCAGCCTCTTTTA GCGCTTTTCCCATGTCGTCCGTATGGACGACCTCTTTGCCCATGTCGCTGAGGCAGAAATTGAAACTGGCATTGCAACCAAAGAAGGAGGAAAAACTGCTGCAGGTCT CGGAGGAATTAGTCATGGAGGCCAAGGCTGCTTTTGAGGATGCTCAGGAGGAAGCCAGAGCGGAGAAGCTCCGAGAAGCACTTCCACCATTAGTGGCAGACAAAGGCA TCGAGGCAGCCGCAGAAGTTGTCTGCGAAGTGGAGGGGCTCCAGGCGGACATCGGAGCAGCATTAGTTGAAACCCCGCGCGGTCACGTAAGGATAATACCTCAAGCAA ATGACCGTATGATCGGACAGTATATCGTTGTCTCGCCAAACTCTGTGCTGAAGAATGCCAAACTCGCACCAGCGCACCCGCTAGCAGATCAGGTTAAGATCATAACAC ACTCCGGAAGATCAGGAAGGTACGCGGTCGAACCATACGACGCTAAAGTACTGATGCCAGCAGGAGGTGCCGTACCATGGCCAGAATTCCTAGCACTGAGTGAGAGCG CCACGTTAGTGTACAACGAAAGAGAGTTTGTGAACCGCAAACTATACCACATTGCCATGCATGGCCCCGCCAAGAATACAGAAGAGGAGCAGTACAAGGTTACAAAGG CAGAGCTTGCAGAAACAGAGTACGTGTTTGACGTGGACAAGAAGCGTTGCGTTAAGAAGGAAGAAGCCTCAGGTCTGGTCCTCTCGGGAGAACTGACCAACCCTCCCT ATCATGAGCTAGCTCTGGAGGGACTGAAGACCCGACCTGCGGTCCCGTACAAGGTCGAAACAATAGGAGTGATAGGCACACCGGGGTCGGGCAAGTCAGCTATTATCA AGTCAACTGTCACGGCACGAGATCTTGTTACCAGCGGAAAGAAAGAAAATTGTCGCGAAATTGAGGCCGACGTGCTAAGACTGAGGGGTATGCAGATTACGTCGAAGA CAGTAGATTCGGTTATGCTCAACGGATGCCACAAAGCCGTAGAAGTGCTGTACGTTGACGAAGCGTTCGCGTGCCACGCAGGAGCACTACTTGCCTTGATTGCTATCG TCAGGCCCCGCAAGAAGGTAGTACTATGCGGAGACCCCATGCAATGCGGATTCTTCAACATGATGCAACTAAAGGTACATTTCAATCACCCTGAAAAAGACATATGCA CCAAGACATTCTACAAGTATATCTCCCGGCGTTGCACACAGCCAGTTACAGCTATTGTATCGACACTGCATTACGATGGAAAGATGAAAACCACGAACCCGTGCAAGA AGAACATTGAAATCGATATTACAGGGGCCACAAAGCCGAAGCCAGGGGATATCATCCTGACATGTTTCCGCGGGTGGGTTAAGCAATTGCAAATCGACTATCCCGGAC ATGAAGTAATGACAGCCGCGGCCTCACAAGGGCTAACCAGAAAAGGAGTGTATGCCGTCCGGCAAAAAGTCAATGAAAACCCACTGTACGCGATCACATCAGAGCATG TGAACGTGTTGCTCACCCGCACTGAGGACAGGCTAGTGTGGAAAACCTTGCAGGGCGACCCATGGATTAAGCAGCCCACTAACATACCTAAAGGAAACTTTCAGGCTA CTATAGAGGACTGGGAAGCTGAACACAAGGGAATAATTGCTGCAATAAACAGCCCCACTCCCCGTGCCAATCCGTTCAGCTGCAAGACCAACGTTTGCTGGGCGAAAG CATTGGAACCGATACTAGCCACGGCCGGTATCGTACTTACCGGTTGCCAGTGGAGCGAACTGTTCCCACAGTTTGCGGATGACAAACCACATTCGGCCATTTACGCCT TAGACGTAATTTGCATTAAGTTTTTCGGCATGGACTTGACAAGCGGACTGTTTTCTAAACAGAGCATCCCACTAACGTACCATCCCGCCGATTCAGCGAGGCCGGTAG CTCATTGGGACAACAGCCCAGGAACCCGCAAGTATGGGTACGATCACGCCATTGCCGCCGAACTCTCCCGTAGATTTCCGGTGTTCCAGCTAGCTGGGAAGGGCACAC AACTTGATTTGCAGACGGGGAGAACCAGAGTTATCTCTGCACAGCATAACCTGGTCCCGGTGAACCGCAATCTTCCTCACGCCTTAGTCCCCGAGTACAAGGAGAAGC AACCCGGCCCGGTCAAAAAATTCTTGAACCAGTTCAAACACCACTCAGTACTTGTGGTATCAGAGGAAAAAATTGAAGCTCCCCGTAAGAGAATCGAATGGATCGCCC CGATTGGCATAGCCGGTGCAGATAAGAACTACAACCTGGCTTTCGGGTTTCCGCCGCAGGCACGGTACGACCTGGTGTTCATCAACATTGGAACTAAATACAGAAACC ACCACTTTCAGCAGTGCGAAGACCATGCGGCGACCTTAAAAACCCTTTCGCGTTCGGCCCTGAATTGCCTTAACCCAGGAGGCACCCTCGTGGTGAAGTCCTATGGCT ACGCCGACCGCAACAGTGAGGACGTAGTCACCGCTCTTGCCAGAAAGTTTGTCAGGGTGTCTGCAGCGAGACCAGATTGTGTCTCAAGCAATACAGAAATGTACCTGA TTTTCCGACAACTAGACAACAGCCGTACACGGCAATTCACCCCGCACCATCTGAATTGCGTGATTTCGTCCGTGTATGAGGGTACAAGAGATGGAGTTGGAGCCGCGC CGTCATACCGCACCAAAAGGGAGAATATTGCTGACTGTCAAGAGGAAGCAGTTGTCAACGCAGCCAATCCGCTGGGTAGACCAGGCGAAGGAGTCTGCCGTGCCATCT ATAAACGTTGGCCGACCAGTTTTACCGATTCAGCCACGGAGACAGGCACCGCAAGAATGACTGTGTGCCTAGGAAAGAAAGTGATCCACGCGGTCGGCCCTGATTTCC GGAAGCACCCAGAAGCAGAAGCCTTGAAATTGCTACAAAACGCCTACCATGCAGTGGCAGACTTAGTAAATGAACATAACATCAAGTCTGTCGCCATTCCACTGCTAT CTACAGGCATTTACGCAGCCGGAAAAGACCGCCTTGAAGTATCACTTAACTGCTTGACAACCGCGCTAGACAGAACTGACGCGGACGTAACCATCTATTGCCTGGATA AGAAGTGGAAGGAAAGAATCGACGCGGCACTCCAACTTAAGGAGTCTGTAACAGAGCTGAAGGATGAAGATATGGAGATCGACGATGAGTTAGTATGGATTCATCCAG ACAGTTGCTTGAAGGGAAGAAAGGGATTCAGTACTACAAAAGGAAAATTGTATTCGTACTTCGAAGGCACCAAATTCCATCAAGCAGCAAAAGACATGGCGGAGATAA AGGTCCTGTTCCCTAATGACCAGGAAAGTAATGAACAACTGTGTGCCTACATATTGGGTGAGACCATGGAAGCAATCCGCGAAAAGTGCCCGGTCGACCATAACCCGT CGTCTAGCCCGCCCAAAACGTTGCCGTGCCTTTGCATGTATGCCATGACGCCAGAAAGGGTCCACAGACTTAGAAGCAATAACGTCAAAGAAGTTACAGTATGCTCCT CCACCCCCCTTCCTAAGCACAAAATTAAGAATGTTCAGAAGGTTCAGTGCACGAAAGTAGTCCTGTTTAATCCGCACACTCCCGCATTCGTTCCCGCCCGTAAGTACA TAGAAGTGCCAGAACAGCCTACCGCTCCTCCTGCACAGGCCGAGGAGGCCCCCGAAGTTGTAGCGACACCGTCACCATCTACAGCTGATAACACCTCGCTTGATGTCA CAGACATCTCACTGGATATGGATGACAGTAGCGAAGGCTCACTTTTTTCGAGCTTTAGCGGATCGGACAACTCTATTACTAGTATGGACAGTTGGTCGTCAGGACCTA GTTCACTAGAGATAGTAGACCGAAGGCAGGTGGTGGTGGCTGACGTTCATGCCGTCCAAGAGCCTGCCCCTATTCCACCGCCAAGGCTAAAGAAGATGGCCCGCCTGG CAGCGGCAAGAAAAGAGCCCACTCCACCGGCAAGCAATAGCTCTGAGTCCCTCCACCTCTCTTTTGGTGGGGTATCCATGTCCCTCGGATCAATTTTCGACGGAGAGA CGGCCCGCCAGGCAGCGGTACAACCCCTGGCAACAGGCCCCACGGATGTGCCTATGTCTTTCGGATCGTTTTCCGACGGAGAGATTGATGAGCTGAGCCGCAGAGTAA CTGAGTCCGAACCCGTCCTGTTTGGATCATTTGAACCGGGCGAAGTGAACTCAATTATATCGTCCCGATCAGCCGTATCTTTTCCACTACGCAAGCAGAGACGTAGAC GCAGGAGCAGGAGGACTGAATACTGACTAACCGGGGTAGGTGGGTACATATTTTCGACGGACACAGGCCCTGGGCACTTGCAAAAGAAGTCCGTTCTGCAGAACCAGC TTACAGAACCGACCTTGGAGCGCAATGTCCTGGAAAGAATTCATGCCCCGGTGCTCGACACGTCGAAAGAGGAACAACTCAAACTCAGGTACCAGATGATGCCCACCG AAGCCAACAAAAGTAGGTACCAGTCTCGTAAAGTAGAAAATCAGAAAGCCATAACCACTGAGCGACTACTGTCAGGACTACGACTGTATAACTCTGCCACAGATCAGC CAGAATGCTATAAGATCACCTATCCGAAACCATTGTACTCCAGTAGCGTACCGGCGAACTACTCCGATCCACAGTTCGCTGTAGCTGTCTGTAACAACTATCTGCATG AGAACTATCCGACAGTAGCATCTTATCAGATTACTGACGAGTACGATGCTTACTTGGATATGGTAGACGGGACAGTCGCCTGCCTGGATACTGCAACCTTCTGCCCCG CTAAGCTTAGAAGTTACCCGAAAAAACATGAGTATAGAGCCCCGAATATCCGCAGTGCGGTTCCATCAGCGATGCAGAACACGCTACAAAATGTGCTCATTGCCGCAA CTAAAAGAAATTGCAACGTCACGCAGATGCGTGAACTGCCAACACTGGACTCAGCGACATTCAATGTCGAATGCTTTCGAAAATATGCATGTAATGACGAGTATTGGG AGGAGTTCGCTCGGAAGCCAATTAGGATTACCACTGAGTTTGTCACCGCATATGTAGCTAGACTGAAAGGCCCTAAGGCCGCCGCACTATTTGCAAAGACGTATAATT TGGTCCCATTGCAAGAAGTGCCTATGGATAGATTCGTCATGGACATGAAAAGAGACGTGAAAGTTACACCAGGCACGAAACACACAGAAGAAAGACCGAAAGTACAAG TGATACAAGCCGCAGAACCCCTGGCGACTGCTTACTTATGCGGGATTCACCGGGAATTAGTGCGTAGGCTTACGGCCGTCTTGCTTCCAAACATTCACACGCTTTTTG ACATGTCGGCGGAGGATTTTGATGCAATCATAGCAGAACACTTCAAGCAAGGCGACCCGGTACTGGAGACGGATATCGCATCATTCGACAAAAGCCAAGACGACGCTA TGGCGTTAACCGGTCTGATGATCTTGGAGGACCTGGGTGTGGATCAACCACTACTCGACTTGATCGAGTGCGCCTTTGGAGAAATATCATCCACCCATCTACCTACGG GTACTCGTTTTAAATTCGGGGCGATGATGAAATCCGGAATGTTCCTCACACTTTTTGTCAACACAGTTTTGAATGTCGTTATCGCCAGCAGAGTACTAGAAGAGCGGC TTAAAACGTCCAGATGTGCAGCGTTCATTGGCGACGACAACATCATACATGGAGTAGTATCTGACAAAGAAATGGCTGAGAGGTGCGCCACCTGGCTCAACATGGAGG TTAAGATCATCGACGCAGTCATCGGTGAGAGACCACCTTACTTCTGCGGCGGATTTATCTTGCAAGATTCGGTTACTTCCACAGCGTGCCGCGTGGCGGATCCCCTGA AAAGGCTGTTTAAGTTGGGTAAACCGCTCCCAGCCGACGACGAGCAAGACGAAGACAGAAGACGCGCTCTGCTAGATGAAACAAAGGCGTGGTTTAGAGTAGGTATAA CAGGCACTTTAGCAGTGGCCGTGACGACCCGGTATGAGGTAGACAATATTACACCTGTCCTACTGGCATTGAGAACTTTTGCCCAGAGCAAAAGAGCATTCCAAGCCA TCAGAGGGGAAATTAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAGACCACCATGGTGAGCA AGGGCGAGGAGAATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGG GCCGCCCCTACGAGGCCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGG TCTACATTAAGCACCCAGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAGGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCATTATTCACG TTAACCAGGACTCCTCCCTGCAGGACGGCGTGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCT GGGAGGCCTCCGAGGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGAGCGAGATCAAGAAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGCCGCCGAGGTCAAGA CCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACATCGTCGACATCAAGTTGGACATCGTGTCCCACAACGAGGACTACACCATCGTGGAACAGTACG AACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCCATGGCCGGGTGGAACGCCTACATCGACA GCCTTATGGCGGACGGGACCTGTCAGGACGCGGCCATCGTAGGCTACAAGGACTCGCCCTCCGTCTGGGCCGCCGTCCCCGGGAAGACCTTCGTTAGCATTACGCCAG CTGAGGTTGGTGTCCTGGTAGGCAAAGACCGGTCAAGTTTTTTCGTCAATGGGCTGACACTTGGGGGCCAGAAATGTTCTGTGATCCGGGACTCACTGCTGCAAGACG GGGAATTTACAATGGATCTTCGTACCAAGAGCACCGGAGGAGCCCCCACCTTCAATGTCACTGTCACCATGACTGCCAAGACGCTAGTCCTGCTGATGGGCAAAGAAG GTGTCCACGGTGGTTTGATCAACAAGAAATGTTATGAAATGGCCTCTCACCTGCGGCGTTCCCAGTACTAAACCGCATGCAGGCCTTAAAGCATCTCTACGGTGGTCC TAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAGTAGAGGCGCACCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTG CCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC ACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTCTCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTC AAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATCTTCTTCGAGGATGACGGCAACTACAAGTCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTG AATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTACAACTACAACGCCCACAATGTGTACATCATGACCGACAAG GCCAAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCT GTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTACTTCGGCTTCGTGACCGCCGCCGCC ATCACCCACGGCATGGATGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCC GGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGC GACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTAC AATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTC AACACCCCAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGCATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTG CCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACTGACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGC TTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTCC CTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCC TGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCT GGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGC TCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCTTCTAGGGGCCCAAT GATCCGACCAGCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAACACTAAAAA CTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGTTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATTTAGCGGACGCCAAAAACTCAATGTATTTTTG AGGAAGCGTGGTGCATAATGCCACGCAGGTTTTGCATAATTTTTTTTTTTTTTTTTATTAATCAACAAAATTTTGTTTTTAACATTTCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAGGGGAATTCCTCGATAATTAAGCGGCCGCTTAATTAATCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATG TGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTA TGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC TGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAA AGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGG AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC CTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA ACGCCAGCAACGCGAGCTCGTATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGGGACACTATAG

TCTAGA

CCACC

ATGGTGAGCAAGGGCGAGGAGAATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGC GAGGGCGAGGGCCGCCCCTACGAGGCCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTAC GGCTCCAAGGTCTACATTAAGCACCCAGCCGACATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAGGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC ATTATTCACGTTAACCAGGACTCCTCCCTGCAGGACGGCGTGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAG ACCATGGGCTGGGAGGCCTCCGAGGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGAGCGAGATCAAGAAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGCCGCC GAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACATCGTCGACATCAAGTTGGACATCGTGTCCCACAACGAGGACTACACCATCGTG GAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCCATGGCCGGGTGGAACGCC TACATCGACAGCCTTATGGCGGACGGGACCTGTCAGGACGCGGCCATCGTAGGCTACAAGGACTCGCCCTCCGTCTGGGCCGCCGTCCCCGGGAAGACCTTCGTTAGC ATTACGCCAGCTGAGGTTGGTGTCCTGGTAGGCAAAGACCGGTCAAGTTTTTTCGTCAATGGGCTGACACTTGGGGGCCAGAAATGTTCTGTGATCCGGGACTCACTG CTGCAAGACGGGGAATTTACAATGGATCTTCGTACCAAGAGCACCGGAGGAGCCCCCACCTTCAATGTCACTGTCACCATGACTGCCAAGACGCTAGTCCTGCTGATG GGCAAAGAAGGTGTCCACGGTGGTTTGATCAACAAGAAATGTTATGAAATGGCCTCTCACCTGCGGCGTTCCCAGTACTAA

ACC

GCATGC

AGGCCT

TAAAGCATCTCTACGGTGGTCCTAAATAGTCAGCATAGTACATTTCATCTGACTAATACCACAACACCACCACCTCTAG

TAGAGGCGCACC

ATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATCGTGCCCATCCTGATCGAGCTGAATGGCGATGTGAATGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCCTGGCCCACCCTGGTGACCACCCTGAGCTACGGCGTGCAGTGCTTC TCACGCTACCCCGATCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACATCCAGGAGCGCACCATCTTCTTCGAGGATGACGGCAACTACAAG TCGCGCGCCGAGGTGAAGTTCGAGGGCGATACCCTGGTGAATCGCATCGAGCTGACCGGCACCGATTTCAAGGAGGATGGCAACATCCTGGGCAATAAGATGGAGTAC AACTACAACGCCCACAATGTGTACATCATGACCGACAAGGCCAAGAATGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGATGGCAGCGTGCAGCTGGCC GACCACTACCAGCAGAATACCCCCATCGGCGATGGCCCTGTGCTGCTGCCCGATAACCACTACCTGTCCACCCAGAGCGCCCTGTCCAAGGACCCCAACGAGAAGCGC GATCACATGATCTACTTCGGCTTCGTGACCGCCGCCGCCATCACCCACGGCATGGATGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTATGGATGATGATATCGCC GCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGC GTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAAC TGGGACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCC AACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACACCCCAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGC ATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTGCCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACT GACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGAC TTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTCCCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGCCCT GAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTAC GCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAGATCATT GCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGCGGCTCCATCCTGGCCTCGCTGTCCACCTTCCAGCAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGC CCCTCCATCGTCCACCGCAAATGCTTCTAG


GGGCCCAATGATCCGACCAGCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAA CACTAAAAACTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGTTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATTTAGCGGACGCCAAAAACTCAAT GTATTTTTGAGGAAGCGTGGTGCATAATGCCACGCAGGTTTTGCATAATTTTTTTTTTTTTTTTTATTAATCAACAAAATTTTGTTTTTAACATTTC


AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA


GGGGAATTCCTCGATAATTAAGCGGCCGCTTAATTAATCGAGGGGAATTAATTCTTGAAGACGAAAGGGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCT ATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCG GTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTT TTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCA ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG GTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT GAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAAC GCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG AGCTCGTATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGGGACACTATAG