LOCUS       sequence-369047-        7647 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Multisite destination vector for ascidian electroporation of an 
            AttB1/B2 recombined fusion N-ter Venus-gene of interest 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 7647)
  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 7647)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-venus
FEATURES             Location/Qualifiers
     source          1..7647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    34..157
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(586..891)
                     /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(1236..1895)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1896..1998)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2176..2299)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2327..2343)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2327..2343)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2373..2497
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2522..2527,2528..2545,2546..2552)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2606..3265
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3607..3912
                     /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(3953..4077)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(4091..4093,4094..4096,4097..4609)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(4136..4157)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4397..4416)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4744..4765
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5109..5243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5146..5165)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5200..5219
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5279..5298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5280..5298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5385..5402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5556..6144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5636..5655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6315..7106,7107..7175))
                     /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     6938..6957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7176..7280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7348..7366
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7404..7426)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7526..7545
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7626..7644
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7626..7643
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     join(7647,1..16)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 cgaggtcgac ggtatcgata agcttgatgg ggacaagttt gtataataaa gttgaacgag
       61 aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
      121 cataatactg taaaacacaa catatccagt cactatgaat caactactta gatggtatta
      181 gtgacctgta gtcgaccgac agccttccaa atgttcttcg ggtgatgctg ccaacttagt
      241 cgaccgacag ccttccaaat gttcttctca aacggaatcg tcgtatccag cctactcgct
      301 attgtcctca atgccgtatt aaatcataaa aagaaataag aaaaagaggt gcgagcctct
      361 tttttgtgtg acaaaataaa aacatctacc tattcatata cgctagtgtc atagtcctga
      421 aaatcatctg catcaagaac aatttcacaa ctcttatact tttctcttac aagtcgttcg
      481 gcttcatctg gattttcagc ctctatactt actaaacgtg ataaagtttc tgtaatttct
      541 actgtatcga cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa
      601 catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc
      661 cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc
      721 cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact
      781 ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac
      841 aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc
      901 ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc
      961 cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg
     1021 ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc
     1081 gctgtcaact gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg
     1141 gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt
     1201 tatctggctt ttagtaagcc ggatccacgc gnnntttacg ccccgccctg ccactcatcg
     1261 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     1321 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     1381 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     1441 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     1501 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     1561 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     1621 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat
     1681 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     1741 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     1801 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     1861 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     1921 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     1981 cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta
     2041 tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat
     2101 tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc actaatacca
     2161 tctaagtagt tgattcatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     2221 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     2281 acttttgtat acaaagtggt ccccatcgaa ttcctgcagc ccgggggatc cactagttct
     2341 agaggatcca tttaaatagg cctttgttta tcacaagttt gtacaaaaaa gctgaacgag
     2401 aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
     2461 cataatactg taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg
     2521 ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccgtcgag
     2581 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     2641 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     2701 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     2761 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     2821 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     2881 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     2941 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     3001 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     3061 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     3121 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     3181 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     3241 cgatgagtgg cagggcgggg cgtaaacgcg tggatccggc ttactaaaag ccagataaca
     3301 gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg
     3361 aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac
     3421 agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca
     3481 tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga
     3541 tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct
     3601 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     3661 gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt
     3721 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     3781 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     3841 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     3901 tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga
     3961 ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa
     4021 tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggtgat
     4081 aaacttagat atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     4141 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     4201 tgccacctac ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc
     4261 ctggcccacc ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga
     4321 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     4381 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     4441 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     4501 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa
     4561 gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt
     4621 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4681 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     4741 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actcatcgga attccagctg
     4801 agcgccggtc gctaccatta ccagttggtc tggtgtcaaa aataataata accgggcagg
     4861 ccatgtctgc ccgtatttcg cgtaaggaaa tccattatgt actatttaaa aaacacaaac
     4921 ttttggatgt tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg
     4981 tttttcccga tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt
     5041 tttgccgcta tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac
     5101 aaactcggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5161 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5221 atgtatctta tcatgtctgg atcgacaaag tcaaagcggc catcagatct gccggtctcc
     5281 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     5341 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5401 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5461 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5521 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5581 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5641 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5701 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5761 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5821 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5881 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5941 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6001 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6061 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6121 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6181 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6241 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6301 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6361 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6421 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6481 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6541 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6601 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6661 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6721 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6781 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6841 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6901 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6961 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7021 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7081 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7141 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7201 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7261 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7321 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     7381 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     7441 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     7501 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     7561 atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
     7621 atacgattta ggtgacacta tagaact
//
