LOCUS       sequence-225195-        5812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent vector with zebrafish pax3a enhancer 5; Tol2 version.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5812)
  AUTHORS   Chong-Morrison V, Mayes S, Simoes FC, Senanayake U, Carroll DS, 
            Riley PR, Wilson SW, Sauka-Spengler T
  TITLE     Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in 
            zebrafish.
  JOURNAL   Biol Open. 2023 Jun 15;12(6):bio059995. doi: 10.1242/bio.059995. 
            Epub 2023 Jun 27.
  PUBMED    37367831
REFERENCE   2  (bases 1 to 5812)
  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: "Biol 
            Open."; date: "2023-06-15"; volume: "12(6):bio059995. doi"; pages: "
            10.1242/bio.059995. Epub 2023 Jun 27"
COMMENT     Sequence Label: pVC-Tol2-Pax3a_enh5-E1b:eGFP-Tol2
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      353..941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     842..861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1095..1112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1229..1250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1265..1270,1271..1288,1289..1295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1303..1319
                     /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)."
     primer_bind     1308..1330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1327..1343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1327..1343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1362..1382
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1364..1382
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1419..1435
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1419..1435
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(1469..1480,1481..1668)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     intron          2437..3009
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      3083..3092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3089..3091,3092..3094,3095..3808)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3089..3091,3092..3094,3095..3805)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3134..3155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3395..3414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3742..3763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4018..4099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4055..4074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(4183..4320,4321..4332)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4390..4407)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4390..4406)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4399..4421)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4548..5003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4635..4654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4845..4866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5029..5133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5134..5202,5203..5812,1..182)
                     /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     complement(5352..5371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
       61 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      181 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      241 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      301 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      361 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      421 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      481 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
      541 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      601 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      661 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      721 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      781 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      841 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      901 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      961 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1021 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1081 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     1141 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1201 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1261 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1321 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     1381 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg
     1441 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt
     1501 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa
     1561 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct
     1621 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttgg gcccggctcg
     1681 agccacacaa ttgtcccaac acaaatcgat taaattaaaa taatcgtttt atttttaatc
     1741 cacttcaatt tgtaaaaaca attaattaaa aaagtagttt gaacaagcag caaaagtaat
     1801 attttcagtg tacaaagcgc caaaagcact gcagataatt tcttatttgt ttttgcgccc
     1861 tcttgcggac agaagaatat tgcagggaaa gccgctttga gaggtgcgtg aatcttcatg
     1921 tctctccggc tttaccacct gcagagagga gcgctactgg tagccatgat cacctgagag
     1981 caataatgcc ggagctcagg ccccggggcc aaggttcacc gcgggggcaa taaactcctg
     2041 tccccctgcc atcatgtaaa gacattaccg tgccctgcgg ccctcacaac aaacactcca
     2101 tttccaaagg ctctctgccg cttactgggt cgctttattt accctgtgtc atgcaaaagt
     2161 cttaatttcc tttgaatccc agtgctgctg tattgccttg agttacttac attcattacc
     2221 accgtttaat tagtaccatc aaacgcttca cggtttacag tggctcaatg tgacattagt
     2281 cgagactgtt aaactcatta gcaaatactg ataagggggt aaaggtgaga tggctgagat
     2341 ctctcgactc tagagggtat ataatggatc ccatcgcgtc tcagcctcac tttgagctcc
     2401 tccacacgaa tttcgaccga tcctgagaac ttcagggtga gtttggggac ccttgattgt
     2461 tctttctttt tcgctattgt aaaattcatg ttatatggag ggggcaaagt tttcagggtg
     2521 ttgtttagaa tgggaagatg tcccttgtat caccatggac cctcatgata attttgtttc
     2581 tttcactttc tactctgttg acaaccattg tctcctctta ttttcttttc attttctgta
     2641 actttttcgt taaactttag cttgcatttg taacgaattt ttaaattcac ttttgtttat
     2701 ttgtcagatt gtaagtactt tctctaatca cttttttttc aaggcaatca gggtatatta
     2761 tattgtactt cagcacagtt ttagagaaca attgttataa ttaaatgata aggtagaata
     2821 tttctgcata taaattctgg ctggcgtgga aatattctta ttggtagaaa caactacacc
     2881 ctggtcatca tcctgccttt ctctttatgg ttacaatgat atacactgtt tgagatgagg
     2941 ataaaatact ctgagtccaa accgggcccc tctgctaacc atgttcatgc cttcttctct
     3001 ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa
     3061 ttcctcgacg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     3121 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3181 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3241 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     3301 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3361 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3421 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3481 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3541 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3601 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3661 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3721 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3781 tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagaattc caactgagcg
     3841 ccggtcgcta ccattaccaa cttgtctggt gtcaaaaata ataggcctac tagtaccgga
     3901 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     3961 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     4021 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4081 aaagcatttt tttcactgcc aattgcattc attttatgtt tcaggttcag ggggaggtgt
     4141 gggaggtttt ttgatatccc gggtttaaac gcatgcagat ctaatactca agtacaattt
     4201 taatggagta cttttttact tttactcaag taagattcta gccagatact tttactttta
     4261 attgagtaaa attttcccta agtacttgta ctttcacttg agtaaaattt ttgagtactt
     4321 tttacacctc tgtcaagaac catatgccgg tacccaatcg ccctatagga cgtcgtatta
     4381 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4441 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4501 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     4561 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4621 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4681 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4741 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4801 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4861 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4921 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4981 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5101 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5641 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ct
//
