LOCUS       sequence-197275-        5337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent vector for screening enhancer elements in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5337)
  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 5337)
  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-Ds-E1b:eGFP-Ds
FEATURES             Location/Qualifiers
     source          1..5337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(32..460)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(142..163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     354..373
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     587..609
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     601..618
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     602..618
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     628..647
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        628..646
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1054..1626
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1700..1709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1706..1708,1709..1711,1712..2425)
                     /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(1706..1708,1709..1711,1712..2422)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1751..1772)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2012..2031)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2359..2380
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2635..2716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2672..2691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2726..2745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3151..3171)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3151..3169)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3190..3206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3190..3206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3203..3225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3214..3230
                     /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(3238..3244,3245..3262,3263..3268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3283..3304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3421..3438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3592..4180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3672..3691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4351..5142,5143..5211))
                     /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     4974..4993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5212..5316)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
       61 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      121 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      181 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      241 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      301 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      361 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      421 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
      481 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      541 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      601 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc
      661 cgacgcaggg atgaaagtag gatgggaaaa tcccgtaccg accgttatcg tataaccgat
      721 tttgttagtt ttatcccgat cgatttcgaa cccgaggtaa aaaacgaaaa cggaacggaa
      781 acgggatata caaaacggta aacggaaacg gaaacggtag agctagtttc ccgaccgttt
      841 caccgggatc ccgtttttaa tcgggatgat cccgtttcgt taccgtattt tctaattcgg
      901 gatgactgca acagatccct cgactcgagt ttacgtaccg ctagcaagct tgcggcgata
      961 tcgactctag agggtatata atggatccca tcgcgtctca gcctcacttt gagctcctcc
     1021 acacgaattt cgaccgatcc tgagaacttc agggtgagtt tggggaccct tgattgttct
     1081 ttctttttcg ctattgtaaa attcatgtta tatggagggg gcaaagtttt cagggtgttg
     1141 tttagaatgg gaagatgtcc cttgtatcac catggaccct catgataatt ttgtttcttt
     1201 cactttctac tctgttgaca accattgtct cctcttattt tcttttcatt ttctgtaact
     1261 ttttcgttaa actttagctt gcatttgtaa cgaattttta aattcacttt tgtttatttg
     1321 tcagattgta agtactttct ctaatcactt ttttttcaag gcaatcaggg tatattatat
     1381 tgtacttcag cacagtttta gagaacaatt gttataatta aatgataagg tagaatattt
     1441 ctgcatataa attctggctg gcgtggaaat attcttattg gtagaaacaa ctacaccctg
     1501 gtcatcatcc tgcctttctc tttatggtta caatgatata cactgtttga gatgaggata
     1561 aaatactctg agtccaaacc gggcccctct gctaaccatg ttcatgcctt cttctctttc
     1621 ctacagctcc tgggcaacgt gctggttgtt gtgctgtctc atcattttgg caaagaattc
     1681 ctcgacggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     1741 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1801 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1861 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     1921 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1981 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2041 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2101 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2161 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2221 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2281 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2341 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2401 cggcatggac gagctgtaca agtaaagcgg ccgcgactct agaattccaa ctgagcgccg
     2461 gtcgctacca ttaccaactt gtctggtgtc aaaaataata ggcctactag taccggatct
     2521 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2581 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2641 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2701 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt agagtagact
     2761 tatatggctt cttatgttag ccaagagccc aagacttatc acttatgtgc tacattaaac
     2821 tatgtgtgct ccagatttat atggatttta tctatgttta attaagactt gtgtttacaa
     2881 ttttttatat ttgtttttaa gttttgaata tatgttttca tgtgtgattt taccgaacaa
     2941 aaataccggt tcccgtccga tttcgacttt aacccgaccg gatcgtatcg gttttcgatt
     3001 accgtattta tcccgttcgt tttcgttacc ggtatatccc gttttcgttt ccgtcccgca
     3061 agttaaatat gaaaatgaaa acggtagagg tattttaccg accgttaccg accgttttca
     3121 tccctagtcg gggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     3181 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3241 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3301 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3361 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3421 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3481 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3541 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3601 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3661 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3721 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3781 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3841 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3901 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3961 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4021 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4081 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4141 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4201 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4261 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4321 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4381 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4441 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4501 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4561 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4621 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4681 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4741 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4801 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4861 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4921 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4981 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5041 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5101 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5161 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5221 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5281 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgcc
//
