LOCUS       sequence-373702-        7636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite destination vector for ascidian electroporation of an 
            AttB1/B2 recombined fusion gene of interest-C-ter CFP under control 
            of an AttB3/B5 recombined reg. sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7636)
  AUTHORS   Roure A, Rothbacher U, Robin F, Kalmar E, Ferone G, Lamy C, Missero 
            C, Mueller F, Lemaire P
  TITLE     A multicassette Gateway vector set for high throughput and 
            comparative analyses in ciona and vertebrate embryos.
  JOURNAL   PLoS One. 2007. 2(9):e916.
  PUBMED    17878951
REFERENCE   2  (bases 1 to 7636)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One. 
            2007. 2(9):e916."
COMMENT     Sequence Label: pDEST-pSP172BSSPE-R3-ccdB/CmR-R5::RfA-CFP
FEATURES             Location/Qualifiers
     source          1..7636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..21
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6..22
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    39..162
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(591..896)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(1216..1899)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     promoter        complement(1900..2002)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2180..2303)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2331..2347)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2331..2347)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2377..2501
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2526..2531,2532..2549,2550..2556)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2610..3269
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3611..3916
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3957..4081)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(4097..4099,4100..4612)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKIRHNIE"
     primer_bind     complement(4139..4160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4400..4419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4747..4768
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5103..5237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5140..5159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5194..5213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5273..5292)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5274..5292)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5379..5396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5550..6138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5630..5649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6309..7100,7101..7169))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6932..6951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7170..7274)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7342..7360
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7398..7420)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7520..7539
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7620..7636,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7620..7636,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaactcgagg tcgacggtat cgataagctt gatggggaca agtttgtata ataaagttga
       61 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
      121 gactacataa tactgtaaaa cacaacatat ccagtcacta tgaatcaact acttagatgg
      181 tattagtgac ctgtagtcga ccgacagcct tccaaatgtt cttcgggtga tgctgccaac
      241 ttagtcgacc gacagccttc caaatgttct tctcaaacgg aatcgtcgta tccagcctac
      301 tcgctattgt cctcaatgcc gtattaaatc ataaaaagaa ataagaaaaa gaggtgcgag
      361 cctctttttt gtgtgacaaa ataaaaacat ctacctattc atatacgcta gtgtcatagt
      421 cctgaaaatc atctgcatca agaacaattt cacaactctt atacttttct cttacaagtc
      481 gttcggcttc atctggattt tcagcctcta tacttactaa acgtgataaa gtttctgtaa
      541 tttctactgt atcgacctgc agactggctg tgtataaggg agcctgacat ttatattccc
      601 cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact
      661 tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat gcgccagctt
      721 tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt
      781 gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca
      841 tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatttca
      901 ccagcccctg ttctcgtcag caaaagagcc gttcatttca ataaaccggg cgacctcagc
      961 catcccttcc tgattttccg ctttccagcg ttcggcacgc agacgacggg cttcattctg
     1021 catggttgtg cttaccagac cggagatatt gacatcatat atgccttgag caactgatag
     1081 ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag catacctctt tttgacatac
     1141 ttcgggtata catatcagta tatattctta taccgcaaaa atcagcgcgc aaatacgcat
     1201 actgttatct ggcttttagt aagccggatc cacgcggcgt ttacgccccc cctgccactc
     1261 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     1321 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     1381 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     1441 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     1501 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     1561 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     1621 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     1681 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     1741 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     1801 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     1861 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     1921 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
     1981 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
     2041 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
     2101 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta gtcgactaca ggtcactaat
     2161 accatctaag tagttgattc atagtgactg gatatgttgt gttttacagt attatgtagt
     2221 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     2281 ttcaactttt gtatacaaag tggtccccat cgaattcctg cagcccgggg gatccactag
     2341 ttctagagga tccatttaaa taggcctttg tttatcacaa gtttgtacaa aaaagctgaa
     2401 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     2461 actacataat actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc
     2521 caggctttac actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt
     2581 cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg
     2641 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
     2701 gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa
     2761 ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc
     2821 cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt
     2881 gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg
     2941 acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc
     3001 tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg
     3061 tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt
     3121 tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
     3181 ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
     3241 actgcgatga gtggcagggc ggggcgtaaa cgcgtggatc cggcttacta aaagccagat
     3301 aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata
     3361 cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag
     3421 cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca
     3481 accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa
     3541 gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg
     3601 ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     3661 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     3721 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     3781 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     3841 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     3901 gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     3961 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     4021 ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg
     4081 tgataaactt agattggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4141 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     4201 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     4261 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc
     4321 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     4381 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     4441 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     4501 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga
     4561 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag
     4621 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     4681 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     4741 cgatcacatg gtcctgctgg agttcgtgac cgccgccgat cggaattcca gctgagcgcc
     4801 ggtcgctacc attaccagtt ggtctggtgt caaaaataat aataaccggg caggccatgt
     4861 ctgcccgtat ttcgcgtaag gaaatccatt atgtactatt taaaaaacac aaacttttgg
     4921 atgttcggtt tattcttttt cttttacttt tttatcatgg gagcctactt cccgtttttc
     4981 ccgatttggc tacatgacat caaccatatc agcaaaagtg atacgggtat tatttttgcc
     5041 gctatttctc tgttctcgct attattccaa ccgctgtttg gtctgctttc tgacaaactc
     5101 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5161 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5221 cttatcatgt ctggatcgac aaagtcaaag cggccatcag atctgccggt ctccctatag
     5281 tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg
     5341 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6181 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6241 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6301 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6361 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6421 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6481 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6541 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6601 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6661 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6721 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6781 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6841 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6901 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6961 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7021 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7081 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7141 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7261 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     7321 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     7381 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     7441 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     7501 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac
     7561 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     7621 tttaggtgac actata
//
