LOCUS       sequence-373849-        7677 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 Venus 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 7677)
  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 7677)
  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::venus-RfA
FEATURES             Location/Qualifiers
     source          1..7677
                     /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"
     CDS             join(2373..2375,2376..2378,2379..3089)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2418..2439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2679..2698)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3026..3047
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3102..3226
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3251..3256,3257..3274,3275..3281)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3335..3994
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             4336..4641
                     /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(4682..4806)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    5144..5278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5181..5200)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5235..5254
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5314..5333)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5315..5333)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5420..5437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5591..6179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5671..5690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6350..7141,7142..7210))
                     /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     6973..6992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7211..7315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7383..7401
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7439..7461)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7561..7580
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7661..7677,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7661..7677,1)
                     /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 taggcagaaa aaatggtgag caagggcgag gagctgttca
     2401 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2461 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca
     2521 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc
     2581 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2641 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2701 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2761 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2821 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     2881 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2941 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgagca
     3001 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3061 tcactctcgg catggacgag ctgtacaagc ctttgtttat cacaagtttg tacaaaaaag
     3121 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     3181 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
     3241 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     3301 tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     3361 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     3421 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     3481 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     3541 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     3601 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     3661 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     3721 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     3781 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     3841 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     3901 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     3961 acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct tactaaaagc
     4021 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     4081 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt
     4141 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     4201 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     4261 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     4321 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     4381 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     4441 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     4501 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     4561 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     4621 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga
     4681 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     4741 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     4801 agtggtgata aacttagata tcggaattcc agctgagcgc cggtcgctac cattaccagt
     4861 tggtctggtg tcaaaaataa taataaccgg gcaggccatg tctgcccgta tttcgcgtaa
     4921 ggaaatccat tatgtactat ttaaaaaaca caaacttttg gatgttcggt ttattctttt
     4981 tcttttactt ttttatcatg ggagcctact tcccgttttt cccgatttgg ctacatgaca
     5041 tcaaccatat cagcaaaagt gatacgggta ttatttttgc cgctatttct ctgttctcgc
     5101 tattattcca accgctgttt ggtctgcttt ctgacaaact cggaacttgt ttattgcagc
     5161 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5221 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcga
     5281 caaagtcaaa gcggccatca gatctgccgg tctccctata gtgagtcgta ttaatttcga
     5341 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5401 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5461 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5521 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5581 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5641 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5701 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5761 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5821 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5881 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5941 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6001 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6061 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6121 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6181 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6241 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6301 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6361 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6421 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6481 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6541 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6601 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6661 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6721 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6781 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6841 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6901 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6961 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7021 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7081 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7141 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7201 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7261 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7321 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     7381 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     7441 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     7501 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     7561 cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg
     7621 gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga cactata
//
