Product Data Sheet

Recovery

1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 ^o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies

. 载体描述：

pCOLADuet-1 is designed for the coexpression of two target genes from a single plasmid. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). MCS-1 encodes the six-amino acid His•Tag sequence for the creation of a N-terminal fusion and MCS2 encodes the 15 amino acid S•Tag™ peptide after the last restriction site for the creation of a C-terminal fusion if desired. Genes inserted into MCS-1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer. Genes inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer. The vector has the COLA replicon from ColA(1) and the kanamycin resistance gene. This vector can be transformed into the same cell with plasmids containing compatible origins of replication and drug resistance genes for coexpression of upto8target genes.

载体序列：

   1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
   61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
  121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
  181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
  241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
  301 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
  361 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
  421 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
  481 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
  541 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
  601 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt
  661 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
  721 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
  781 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
  841 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
  901 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
  961 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
 1021 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
 1081 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca
 1141 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
 1201 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
 1261 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
 1321 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
 1381 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
 1441 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
 1501 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt
 1561 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
 1621 tgaatgtatt tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg
 1681 ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct
 1741 ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
 1801 aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc
 1861 cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt
 1921 gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg
 1981 cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact
 2041 ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt
 2101 ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag
 2161 ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg
 2221 gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca agaagatcct
 2281 ttacggattc ccgacaccat cactctagat ttcagtgcaa tttatctctt caaatgtagc
 2341 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
 2401 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
 2461 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
 2521 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
 2581 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
 2641 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
 2701 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
 2761 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
 2821 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
 2881 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
 2941 tatcggctga atttgat