LOCUS       sequence-417682-        7940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mVenus-tagged Apollo-NADP+ in zebrafish pancreatic beta 
            cells using a zebrafish insulin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Bui CV, Boswell CW, Ciruna B, Rocheleau JV
  TITLE     Apollo-NADP(+) reveals in vivo adaptation of NADPH/NADP(+) 
            metabolism in electrically activated pancreatic beta cells.
  JOURNAL   Sci Adv. 2023 Oct 6;9(40):eadi8317. doi: 10.1126/sciadv.adi8317. 
            Epub 2023 Oct 4.
  PUBMED    37792934
REFERENCE   2  (bases 1 to 7940)
  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: "Sci Adv."; 
            date: "2023-10-6"; volume: "9(40):eadi8317. doi"; pages: " 
            10.1126/sciadv.adi8317. Epub 2023 Oct 4"
COMMENT     Sequence Label: pDestTol2pA ins:Apollo-NADP+ mVenus
FEATURES             Location/Qualifiers
     source          1..7940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(124..712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(204..223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(883..1674,1675..1743))
                     /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     1506..1525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1744..1848)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2011..2032)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(2381..2392,2393..2555)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     2928..2944
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2964..2984
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    4141..4165
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      4167..4176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5727..5736
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5733..5735,5736..5738,5739..6452)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5778..5799)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6039..6058)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6386..6407
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(6481..6505)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6507..6641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(6704..6724)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(6732..6748)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6764..6898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(7337..7524,7525..7536)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(7570..7586)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7570..7586)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(7623..7643)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7623..7641)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7662..7678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7675..7697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7686..7702
                     /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(7710..7716,7717..7734,7735..7740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7755..7776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7893..7910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
       61 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      121 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      181 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      241 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      301 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      361 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      421 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      481 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      541 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      601 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      661 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      721 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aaggcatttt
      781 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      841 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      901 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
      961 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1021 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1081 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1141 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1201 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1261 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1321 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1381 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1441 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1501 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1561 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1621 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1681 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1741 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1801 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1861 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     1921 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     1981 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2041 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccacgctgg
     2101 ctaagaactc atcagcctcc ccggtccatc tacccacgta ccaatgcacc aattggccac
     2161 aatgacggct actacatggt gccattcctt cctctttata ggaatggaga ctacctcctg
     2221 tccaacaatg ctcttggata cgagtacgcc tacctgttgg acccaggtca ttgcacaaca
     2281 ccagaaatgc cctctgatct gcaaaagacg tgaatatctg ttcagacacc catatccact
     2341 ctgttccaca caggtcagag gtttgtccag gagttcttga cagaggtgta aaaagtactc
     2401 aaaaatttta ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta
     2461 aaagtatctg gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta
     2521 cttgagtatt aaggaagtaa aagtaaaagc aagaaagaaa actagagatt cttgtttaag
     2581 cttttaatct caaaaaacat taaatgaaat gcatacaagg ttttatcctg ctttagaact
     2641 gtttgtattt aattatcaaa ctataagaca gacaatctaa tgccagtaca cgctactcaa
     2701 agttgtaaaa cctcagattt aacttcagta gaagctgatt ctcaaaattg ttagtgtcaa
     2761 gcctagctct tttggggctg aaaagcaatc ctgcagtgct gaaaagcctc tcacaggcag
     2821 ccgatgcggg aagaggtgta ttagtcttga tagagaggct gcaaatagca ggaaacgtga
     2881 gcagagactc cctggtgtct gaaacacagg ccagatgggc cctcgagcag gaaacagcta
     2941 tgaccatgat tacgccaagc tatcaacttt gtatagaaaa gttgtagagc tcacgcgtaa
     3001 gctttgtttg taccataata agctcattat ataatattta aaatgatact gcatcattaa
     3061 ttgatatgtt ttctagttat ttttaaatat tagcaactaa attaggaaat tatccatagt
     3121 aaatttaatg taatattatt tgttatattt aacttcagcc cacagtctag tttagttgtt
     3181 ggtcccttat gagaaaaaat ttgggcacct ctgctctagt gcttgtaaac atttttgagt
     3241 atctatagtt gaacatgaaa gcattttttt tttttttttt ttaagaaaat gaagaaagct
     3301 aaaaacctgt atccactgac ttccattaaa aaaagcaaat actagggatg tctatggtgt
     3361 cttcttttgt gttcaacaaa taagcagaaa gtaactgctg acagaacatt acattttgtg
     3421 tgaatcccct tatattgtgt taaaaactgt agagaaatca tctaataata agaaattatc
     3481 aagaaataaa cagaaaatga tctaatagta tactataaaa tctttcaaaa tccaggccac
     3541 taccatatga ctaaaaccac acatattatg ctttaaaaaa agtcacaatg aagacgtcac
     3601 aaacgttcct gaagactaat gactcgtcta accccatctt accttctgct gtttcactca
     3661 aagaaaagcc accagctgtc tctgtgatct gctcattctg tgttaacttg tgttctcaac
     3721 aattaacaat taaccaaaag cctctggagc tgcgtgagca tcacgatgtg tttgtcttct
     3781 gtgagcgtgg cagtgtccac gaagtcaatg tgaattggtt ggccaaacac agctaatgtg
     3841 gcatgtgtga ttggacagcc acttaaaagt gttaaagtgt gtttacgcac aggattggtt
     3901 ttccgctgaa tcaggcaaaa ccagcagaac cgatctaaac tcgagtataa aagtgagtag
     3961 gagatgctat tcgtcccaaa acatctccac caccatctcc accattcctc gcctctgctt
     4021 cgagaacagg tgagtgtcga gcgggatggt aaatctacag agaatgcgga gtgtagcttg
     4081 tgtacatgtt tttgattaac agagattgta tgtgtgtgtt tgtgtcagtg tgacggatcc
     4141 gcaagtttgt acaaaaaagc aggcttgcca ccatggcaga gcaggtggcc ctgagccgga
     4201 cccaggtgtg cgggatcctg cgggaagagc ttttccaggg cgatgccttc catcagtcgg
     4261 atacacacat attcatcatc atgggtgcag cgggtgacct ggccaagaag aagatctacc
     4321 ccaccatctg gtggctgttc cgggatggcc ttctgcccga aaacaccttc atcgtgggct
     4381 atgctcagtc ccgcctcaca gtggctgaca tccgcaaaca gagtgagccc ttcttcaagg
     4441 ccaccccaga ggagaagctc aagctggagg acttctttgc ccgcaactcc tatgtggctg
     4501 gccagtacga tgatgcagcc tcctaccagc gcctcaacag ccacatgaat gccctccacc
     4561 tggggtcaca ggccaaccgc ctcttctacc tggccttgcc cccgaccgtc tacgaggccg
     4621 tcaccaagaa cattcacgag tcctgcatga gccagatagg ctggaaccgc atcatcgtgg
     4681 agaagccctt cgggagggac ctgcagagct ctgaccggct gtccaaccac atctcctccc
     4741 tgttccgtga ggaccagatc taccgcatcg acaactacct gggcacggag atggtgcaga
     4801 acctcatggt gctgagattt gccaacagga tcttcggccc catctggaac cgggacaaca
     4861 tcgcctgcgt tatcctcacc ttcaaggagc cctttggcac tgagggtcgc gggggctatt
     4921 tcgatgaatt tgggatcatc cgggacgtga tgcagaacca cctactgcag atgctgtgtc
     4981 tggtggccat ggagaagccc gcctccacca actcagatga cgtccgtgat gagaaggtca
     5041 aggtgttgaa atgcatctca gaggtgcagg ccaacaatgt ggtcctgggc cagtacgtgg
     5101 ggaaccccga tggagagggc gaggccacca aagggtacct ggacgacccc acggtgcccc
     5161 gcgggtccac caccgccact tttgcagccg tcgtcctcta tgtggagaat gagaggtggg
     5221 atggggtgcc cttcatcctg cgctgcggca aggccctgaa cgagcgcaag gccgaggtga
     5281 ggctgcagtt ccatgatgtg gccggcgaca tcttccacca gcagtgcaag cgcaacgagc
     5341 tggtgatccg cgtgcagccc aacgaggccg tgtacaccaa gatgatgacc aagaagccgg
     5401 gcatgttctt caaccccgag gagtcggagc tggacctgac ctacggcaac agatacaaga
     5461 acgtgaagct ccctgacgcc tatgagcgcc tcatcctgga cgtcttctgc gggagccaga
     5521 tgcacttcgt gcgcagcgac gagctccgtg aggcctggcg tattttcacc ccactgctgc
     5581 accagattga gctggagaag cccaagccca tcccctatat ttatggcagc cgaggcccca
     5641 cggaggcaga cgagctgatg aagagagtgg gtttccagta tgagggcacc tacaagtggg
     5701 tgaaccccca caagctcaga ccggtcgcca ccatggtgag caagggcgag gagctgttca
     5761 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     5821 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca
     5881 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc
     5941 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     6001 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     6061 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     6121 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     6181 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     6241 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     6301 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc aaactgagca
     6361 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     6421 tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcac
     6481 acccagcttt cttgtacaaa gtgggggatc cagacatgat aagatacatt gatgagtttg
     6541 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     6601 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     6661 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttccaacttt attatacata
     6721 gttgataatt cactggccgt cgttttacgg taccatcgat gatgatccag acatgataag
     6781 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     6841 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     6901 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     6961 aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcctctag atcagatctg
     7021 cgaagatacg gccacgggtg ctcttgatcc tgtggctgat tttggactgt gctgctcgca
     7081 gctgctgatg aatcacatac ttcctccatt ttcttccact gattgactgt tataatttcc
     7141 ctaatttcca ggtcaaggtg ctgtgcattg tggtaataga tgtgacatga cgtcacttcc
     7201 aaaggaccaa tgaacatgtc tgaccaattt catataatgt gaaaacgatt ttcataggca
     7261 gaataaataa catttaaatt aaactgggca tcagcgcaat tcaattggtt tggtaatagc
     7321 aagggaaaat agaatgaagt gatctccaaa aaataagtac tttttgactg taaataaaat
     7381 tgtaaggagt aaaaagtact tttttttcta aaaaaatgta attaagtaaa agtaaaagta
     7441 ttgattttta attgtactca agtaaagtaa aaatccccaa aaataatact taagtacagt
     7501 aatcaagtaa aattactcaa gtactttaca cctctggttc ttgaccccct accttcagca
     7561 agcccagcag atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt
     7621 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     7681 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     7741 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     7801 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7861 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     7921 gttcggctgc ggcgagcggt
//
