LOCUS       sequence-373851-        6993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite destination vector for ascidian electroporation of an 
            AttB1/B2 recombined fusion N-ter HA tag-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 6993)
  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 6993)
  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::HA-RfA
FEATURES             Location/Qualifiers
     source          1..6993
                     /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             2379..2405
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    2418..2542
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2567..2572,2573..2590,2591..2597)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2651..3310
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3652..3957
                     /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(3998..4122)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    4460..4594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4497..4516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4551..4570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4630..4649)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4631..4649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4736..4753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4907..5495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4987..5006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5666..6457,6458..6526))
                     /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     6289..6308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6527..6631)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6699..6717
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6755..6777)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6877..6896
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6977..6993,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6977..6993,1)
                     /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 tagggacaaa aaatggcata cccatacgac gtgccagact
     2401 acgctccttt gtttatcaca agtttgtaca aaaaagctga acgagaaacg taaaatgata
     2461 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     2521 cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc
     2581 ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa
     2641 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     2701 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     2761 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     2821 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat
     2881 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     2941 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     3001 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     3061 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     3121 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     3181 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     3241 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     3301 cggggcgtaa acgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     3361 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     3421 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     3481 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     3541 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     3601 ggtttattga aatgaacggc tcttttgctg acgagaacag ggrctggtga aatgcagttt
     3661 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     3721 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     3781 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     3841 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     3901 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     3961 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     4021 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     4081 atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgataaact tagatatcgg
     4141 aattccagct gagcgccggt cgctaccatt accagttggt ctggtgtcaa aaataataat
     4201 aaccgggcag gccatgtctg cccgtatttc gcgtaaggaa atccattatg tactatttaa
     4261 aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt atcatgggag
     4321 cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc aaaagtgata
     4381 cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg ctgtttggtc
     4441 tgctttctga caaactcgga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4501 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4561 caaactcatc aatgtatctt atcatgtctg gatcgacaaa gtcaaagcgg ccatcagatc
     4621 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4741 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4861 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4921 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4981 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5041 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5101 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5161 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5221 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5281 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5341 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5401 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5461 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5521 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5581 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5641 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5701 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5761 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5821 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5881 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5941 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6001 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6061 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6121 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6181 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6241 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6301 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6361 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6421 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6481 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6541 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6601 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6661 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     6721 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     6781 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     6841 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     6901 gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     6961 gtatcataca catacgattt aggtgacact ata
//
