LOCUS       sequence-417683-        7939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mVenus-tagged R198P mutant of 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 7939)
  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 7939)
  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:R198P mVenus
FEATURES             Location/Qualifiers
     source          1..7939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(51..213,214..225)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     574..595
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        758..862
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(863..931,932..1723)
                     /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(1081..1100)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1894..2482
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2383..2402
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2636..2653
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2770..2791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2806..2811,2812..2829,2830..2836)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2844..2860
                     /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     2849..2871
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2868..2884
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2903..2923
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2905..2923
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2960..2976
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2960..2976
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(3010..3021,3022..3209)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    3648..3782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3798..3814
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3822..3842
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3905..4039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    4041..4065
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4094..4807,4808..4810,4811..4813))
                     /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(4139..4160)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4488..4507
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4747..4768
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4810..4819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6370..6379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(6381..6405)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(7561..7581)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(7601..7617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 atttaatgtt ttttgagatt aaaagcttaa acaagaatct ctagttttct ttcttgcttt
       61 tacttttact tccttaatac tcaagtacaa ttttaatgga gtactttttt acttttactc
      121 aagtaagatt ctagccagat acttttactt ttaattgagt aaaattttcc ctaagtactt
      181 gtactttcac ttgagtaaaa tttttgagta ctttttacac ctctgtcaag aactcctgga
      241 caaacctctg acctgtgtgg aacagagtgg atatgggtgt ctgaacagat attcacgtct
      301 tttgcagatc agagggcatt tctggtgttg tgcaatgacc tgggtccaac aggtaggcgt
      361 actcgtatcc aagagcattg ttggacagga ggtagtctcc attcctataa agaggaagga
      421 atggcaccat gtagtagccg tcattgtggc caattggtgc attggtacgt gggtagatgg
      481 accggggagg ctgatgagtt cttagccagc gtgggccatc gccctgatag acggtttttc
      541 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      601 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      661 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      721 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      781 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      841 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      901 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      961 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1021 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1081 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1141 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1201 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1261 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1321 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1381 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     1441 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1501 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1561 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1621 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1681 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1741 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1801 gatccttttt gataatctca tgaccaaaat gccttaacgt gagttttcgt tccactgagc
     1861 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1921 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1981 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2041 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2101 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2161 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2221 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2281 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2341 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2401 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2461 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2521 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2581 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2641 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2701 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2761 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     2821 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     2881 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
     2941 ccaccgcggt ggcggccgct ctagaactag tggatctgct gggcttgctg aaggtagggg
     3001 gtcaagaacc agaggtgtaa agtacttgag taattttact tgattactgt acttaagtat
     3061 tatttttggg gatttttact ttacttgagt acaattaaaa atcaatactt ttacttttac
     3121 ttaattacat ttttttagaa aaaaaagtac tttttactcc ttacaatttt atttacagtc
     3181 aaaaagtact tattttttgg agatcacttc attctatttt cccttgctat taccaaacca
     3241 attgaattgc gctgatgccc agtttaattt aaatgttatt tattctgcct atgaaaatcg
     3301 ttttcacatt atatgaaatt ggtcagacat gttcattggt cctttggaag tgacgtcatg
     3361 tcacatctat taccacaatg cacagcacct tgacctggaa attagggaaa ttataacagt
     3421 caatcagtgg aagaaaatgg aggaagtatg tgattcatca gcagctgcga gcagcacagt
     3481 ccaaaatcag ccacaggatc aagagcaccc gtggccgtat cttcgcagat ctgatctaga
     3541 ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3601 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3661 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3721 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     3781 tcatcatcga tggtaccgta aaacgacggc cagtgaatta tcaactatgt ataataaagt
     3841 tggaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     3901 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3961 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4021 atcttatcat gtctggatcc cccactttgt acaagaaagc tgggtgtgat tatgatctag
     4081 agtcgcggcc gctttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     4141 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agtttggact
     4201 ggtagctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     4261 gctggtagtg gtcggcgagc tgcacgccgc cgtcctcgat gttgtggcgg atcttgaagt
     4321 tggccttgat gccgttcttc tgcttgtcgg cggtgatata gacgttgtgg ctgttgtagt
     4381 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     4441 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     4501 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     4561 agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa gcactgcagg ccgtagccca
     4621 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atcagcttca
     4681 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     4741 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     4801 ccttgctcac catggtggcg accggtctga gcttgtgggg gttcacccac ttgtaggtgc
     4861 cctcatactg gaaacccact ctcttcatca gctcgtctgc ctccgtgggg cctcggctgc
     4921 cataaatata ggggatgggc ttgggcttct ccagctcaat ctggtgcagc agtggggtga
     4981 aaatacgcca ggcctcacgg agctcgtcgc tgcgcacgaa gtgcatctgg ctcccgcaga
     5041 agacgtccag gatgaggcgc tcataggcgt cagggagctt cacgttcttg tatctgttgc
     5101 cgtaggtcag gtccagctcc gactcctcgg ggttgaagaa catgcccggc ttcttggtca
     5161 tcatcttggt gtacacggcc tcgttgggct gcacgcggat caccagctcg ttgcgcttgc
     5221 actgctggtg gaagatgtcg ccggccacat catggaactg cagcctcacc tcggccttgc
     5281 gctcgttcag ggccttgccg cagcgcagga tgaagggcac cccatcccac ctctcattct
     5341 ccacatagag gacgacggct gcaaaagtgg cggtggtgga cccgcggggc accgtggggt
     5401 cgtccaggta ccctttggtg gcctcgccct ctccatcggg gttccccacg tactggccca
     5461 ggaccacatt gttggcctgc acctctgaga tgcatttcaa caccttgacc ttctcatcac
     5521 ggacgtcatc tgagttggtg gaggcgggct tctccatggc caccagacac agcatctgca
     5581 gtaggtggtt ctgcatcacg tcccggatga tcccaaattc atcgaaatag cccccgcgac
     5641 cctcagtgcc aaagggctcc ttgaaggtga ggataacgca ggcgatgttg tcccggttcc
     5701 agatggggcc gaagatcctg ttggcaaatc tcagcaccat gaggttctgc accatctccg
     5761 tgcccaggta gttgtcgatg gggtagatct ggtcctcacg gaacagggag gagatgtggt
     5821 tggacagccg gtcagagctc tgcaggtccc tcccgaaggg cttctccacg atgatgcggt
     5881 tccagcctat ctggctcatg caggactcgt gaatgttctt ggtgacggcc tcgtagacgg
     5941 tcgggggcaa ggccaggtag aagaggcggt tggcctgtga ccccaggtgg agggcattca
     6001 tgtggctgtt gaggcgctgg taggaggctg catcatcgta ctggccagcc acataggagt
     6061 tgcgggcaaa gaagtcctcc agcttgagct tctcctctgg ggtggccttg aagaagggct
     6121 cactctgttt gcggatgtca gccactgtga ggcgggactg agcatagccc acgatgaagg
     6181 tgttttcggg cagaaggcca tcccggaaca gccaccagat ggtggggtag atcttcttct
     6241 tggccaggtc acccgctgca cccatgatga tgaatatgtg tgtatccgac tgatggaagg
     6301 catcgccctg gaaaagctct tcccgcagga tcccgcacac ctgggtccgg ctcagggcca
     6361 cctgctctgc catggtggca agcctgcttt tttgtacaaa cttgcggatc cgtcacactg
     6421 acacaaacac acacatacaa tctctgttaa tcaaaaacat gtacacaagc tacactccgc
     6481 attctctgta gatttaccat cccgctcgac actcacctgt tctcgaagca gaggcgagga
     6541 atggtggaga tggtggtgga gatgttttgg gacgaatagc atctcctact cacttttata
     6601 ctcgagttta gatcggttct gctggttttg cctgattcag cggaaaacca atcctgtgcg
     6661 taaacacact ttaacacttt taagtggctg tccaatcaca catgccacat tagctgtgtt
     6721 tggccaacca attcacattg acttcgtgga cactgccacg ctcacagaag acaaacacat
     6781 cgtgatgctc acgcagctcc agaggctttt ggttaattgt taattgttga gaacacaagt
     6841 taacacagaa tgagcagatc acagagacag ctggtggctt ttctttgagt gaaacagcag
     6901 aaggtaagat ggggttagac gagtcattag tcttcaggaa cgtttgtgac gtcttcattg
     6961 tgactttttt taaagcataa tatgtgtggt tttagtcata tggtagtggc ctggattttg
     7021 aaagatttta tagtatacta ttagatcatt ttctgtttat ttcttgataa tttcttatta
     7081 ttagatgatt tctctacagt ttttaacaca atataagggg attcacacaa aatgtaatgt
     7141 tctgtcagca gttactttct gcttatttgt tgaacacaaa agaagacacc atagacatcc
     7201 ctagtatttg ctttttttaa tggaagtcag tggatacagg tttttagctt tcttcatttt
     7261 cttaaaaaaa aaaaaaaaaa atgctttcat gttcaactat agatactcaa aaatgtttac
     7321 aagcactaga gcagaggtgc ccaaattttt tctcataagg gaccaacaac taaactagac
     7381 tgtgggctga agttaaatat aacaaataat attacattaa atttactatg gataatttcc
     7441 taatttagtt gctaatattt aaaaataact agaaaacata tcaattaatg atgcagtatc
     7501 attttaaata ttatataatg agcttattat ggtacaaaca aagcttacgc gtgagctcta
     7561 caacttttct atacaaagtt gatagcttgg cgtaatcatg gtcatagctg tttcctgctc
     7621 gagggcccat ctggcctgtg tttcagacac cagggagtct ctgctcacgt ttcctgctat
     7681 ttgcagcctc tctatcaaga ctaatacacc tcttcccgca tcggctgcct gtgagaggct
     7741 tttcagcact gcaggattgc ttttcagccc caaaagagct aggcttgaca ctaacaattt
     7801 tgagaatcag cttctactga agttaaatct gaggttttac aactttgagt agcgtgtact
     7861 ggcattagat tgtctgtctt atagtttgat aattaaatac aaacagttct aaagcaggat
     7921 aaaaccttgt atgcatttc
//
