LOCUS       sequence-122204-        6635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway destination vector to create transgenes for 
            phiC31-mediated transgenesis in zebrafish, contains an attB site and
            alpha-cystallin:YFP (CY) transgenesis marker in backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  AUTHORS   Mosimann C, Puller AC, Lawson KL, Tschopp P, Amsterdam A, Zon LI
  TITLE     Site-directed zebrafish transgenesis into single landing sites with 
            the phiC31 integrase system.
  JOURNAL   Dev Dyn. 2013 Aug;242(8):949-63. doi: 10.1002/dvdy.23989. Epub 2013 
            Jul 3.
  PUBMED    23723152
REFERENCE   2  (bases 1 to 6635)
  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"; doi: 
            "10.1002/dvdy.23989"; journalName: "Dev Dyn"; date: "2013-08"; 
            volume: "242"; issue: "8"; pages: "949-63"
COMMENT     Sequence Label: pDestattB/CY (pCM327)
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     polyA_signal    826..1033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1022..1039
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(1057..1770,1771..1773,1774..1776))
                     /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(1102..1123)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1451..1470
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1710..1731
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2352..2368
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2388..2511
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2552..2857)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2654..2673)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3199..3858)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3773..3792
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3912..3918,3919..3936,3937..3942))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3967..4090)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4098..4114)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4159..4228
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(4447..4465)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4486..4502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4499..4521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4510..4526
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4534..4540,4541..4558,4559..4564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4579..4600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4717..4734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4888..5476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4968..4987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5647..6438,6439..6507))
                     /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     6270..6289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6508..6612)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatctag cggatccggg aattgggaat tcgttaacag
      781 atctgcggcc gcggctcgag tacccaccgc ggtggcggcg atctctcccc agcatgcctg
      841 ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa
      901 tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga
      961 gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa
     1021 ctagaaggca cagtcgaggc tgatcagcgg gccgctttac ttgtacagct cgtccatgcc
     1081 gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     1141 ggggtctttg ctcagggcgg actggtagct caggtagtgg ttgtcgggca gcagcacggg
     1201 gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc cgccgtcctc
     1261 gatgttgtgg cggatcttga agttggcctt gatgccgttc ttctgcttgt cggcggtgat
     1321 atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc
     1381 cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac
     1441 ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta
     1501 gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcgggc
     1561 gaagcactgc aggccgtagc ccagggtggt cacgagggtg ggccagggca cgggcagctt
     1621 gccggtggtg cagatcagct tcagggtcag cttgccgtag gtggcatcgc cctcgccctc
     1681 gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac
     1741 caccccggtg aacagctcct cgcccttgct caccatgttg tcagacctgg taactcctta
     1801 ctgtaactta ttgcacctga tgaacaaaag atgtgaactg aacgcaagta ggaggctaat
     1861 agaggctagt acagtacaaa gatccccaaa tgatgtgtgc cggccttagt ggaggcagac
     1921 ccctggttat atacctgtgc tgcagaaaga ggggcttaca aatggatctc tctggaatgg
     1981 cattatctca tggtgggaac agtgttgtca gcagaatcag cggggtggac tctgacctga
     2041 gaatgacagt ctagcactga atggctcaga caatactctt gtggccaact atcaccggaa
     2101 aataagtgcc tgccaaaaca aaccagacca gatgaagcta catggctgta gaacgtcagt
     2161 atttaatgtg ttgtggccac ttggttcacc agatttaaga gctaattttt ttaaatatca
     2221 cagataatca atcgcttttt ttttttcttg actgaaaagc ttagactgga aaattctttg
     2281 aaaaagtttg cactacagaa aataaataca taacatcctg cactgaatgc acactattaa
     2341 tggtactcga gcaggaaaca gctatgacca tgattacgcc aagctatcaa ctttgtatag
     2401 aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca
     2461 taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat ggtcgacctg
     2521 cagactggct gtgtataagg gagcctgaca tttatattcc ccagaacatc aggttaatgg
     2581 cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga
     2641 ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca
     2701 ccgggtaaag ttcacggggg actttatctg acagcagacg tgcactggcc agggggatca
     2761 ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac agacgataac
     2821 ggctctctct tttataggtg taaaccttaa actgcatttc accagcccct gttctcgtcg
     2881 gcaaaggagc cgttcatttc aataaaccgg gcgacctcag ccatcccttc ctgattttcc
     2941 gctttccagc gttcggcacg cagacgacgg gcttcattct gcatggttgt gcttaccgaa
     3001 ccggagatat tgacatcata tatgccttga gcaactgata gctgtcgctg tcaactgtca
     3061 ctgtaatacg ctgcttcata gcatacctct ttttgacata cttcgggtat acatatcagt
     3121 atatattctt ataccgcaaa aatcagcgcg caaatacgca tactgttatc tggcttttag
     3181 taagccggat cctctagatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     3241 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     3301 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     3361 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     3421 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     3481 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     3541 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     3601 atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat gagcattcat
     3661 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     3721 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     3781 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     3841 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgacg gatcctaact
     3901 caaaatccac acattatacg agccggaagc ataaagtgta aagcctgggg tgcctaatgc
     3961 ggccgccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     4021 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc aactttatta
     4081 tacatagttg ataattcact ggccgtcgtt ttacggtacc tctagagatc cactagtgtc
     4141 gacgatgtag gtcacggtct cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc
     4201 cccgggcgcg tactccacct cacccatctg gtccatcatg atgaacgggt cgaggtggcg
     4261 gtagttgatc ccggcgaacg cgcggcgcac cgggaagccc tcgccctcga aaccgctggg
     4321 cgcggtggtc acggtgagca cgggacgtgc gacggcgtcg gcgggtgcgg atacgcgggg
     4381 cagcgtcagc gggttctcga cggtcacggc gggcatgtcg acactagttc tagccagctt
     4441 ttgttccctt tagtgagggt taatttcgag cttggcgtaa tcatggtcat agctgtttcc
     4501 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4561 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4621 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4681 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4741 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4801 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4861 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4921 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4981 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5041 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5101 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5161 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5221 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5281 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5341 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5401 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5461 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5521 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5581 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5641 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5701 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5761 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5821 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5881 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5941 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6001 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6061 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6121 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6181 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6241 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6301 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6361 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6421 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6481 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6541 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6601 aggggttccg cgcacatttc cccgaaaagt gccac
//
