LOCUS       sequence-211547-        8044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A zebrafish insulin promoter driving the calcium indicator, GCaMP6s.
            Contains red eye marker, and is flanked with I-SceI sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8044)
  AUTHORS   Singh SP, Janjuha S, Hartmann T, Kayisoglu O, Konantz J, Birke S, 
            Murawala P, Alfar EA, Murata K, Eugster A, Tsuji N, Morrissey ER, 
            Brand M, Ninov N
  TITLE     Different developmental histories of beta-cells generate functional 
            and proliferative heterogeneity during islet growth.
  JOURNAL   Nat Commun. 2017 Sep 22;8(1):664. doi: 10.1038/s41467-017-00461-3.
  PUBMED    28939870
REFERENCE   2  (bases 1 to 8044)
  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: "Nat 
            Commun."; date: "2017-09-22"; volume: "8(1):664. doi"; pages: " 
            10.1038/s41467-017-00461-3"
COMMENT     Sequence Label: ins:GCaMP6s; cryaa:RFP
FEATURES             Location/Qualifiers
     source          1..8044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    37..61
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     intron          1256..1828
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1895..1904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1901..1909,1910..1927,1928..1930,1931..1963,1964..1966,
                     1967..1990,1991..2017,2018..2074,2075..2080,2081..2350,
                     2351..2368,2369..2803,2804..2809,2810..3253)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1997..2006
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2287..2308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2414..2435)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2675..2694)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    3494..3615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3531..3550)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3585..3604
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3658..3865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3854..3871
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3889..4599)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4167..4186)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4436..4454
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5183..5203)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5183..5201)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5234..5251)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5234..5250)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5243..5265)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5391..5846)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5528..5549)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5740..5759
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5873..5977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5978..6046,6047..6838)
                     /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(6196..6215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7009..7597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7498..7517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7751..7768
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7885..7906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7921..7926,7927..7944,7945..7951)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7959..7975
                     /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     7964..7986
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7983..7999
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7983..7999
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 taatacgact cactataggg cgaattggag ctcatcacaa gtttgtacaa aaaagcaggc
       61 tccgcggccg cccccttcac cacgcgtaag ctttgtttgt accataataa gctcattata
      121 taatatttaa aatgatactg catcattaat tgatatgttt tctagttatt tttaaatatt
      181 agcaactaaa ttaggaaatt atccatagta aatttaatgt aatattattt gttatattta
      241 acttcagccc acagtctagt ttagttgttg gtcccttatg agaaaaaatt tgggcacctc
      301 tgctctagtg cttgtaaaca tttttgagta tctatagttg aacatgaaag catttttttt
      361 tttttttttt taagaaaatg aagaaagcta aaaacctgta tccactgact tccattaaaa
      421 aaagcaaata ctagggatgt ctatggtgtc ttcttttgtg ttcaacaaat aagcagaaag
      481 taactgctga cagaacatta cattttgtgt gaatcccctt atattgtgtt aaaaactgta
      541 gagaaatcat ctaataataa gaaattatca agaaataaac agaaaatgat ctaatagtat
      601 actataaaat ctttcaaaat ccaggccact accatatgac taaaaccaca catattatgc
      661 tttaaaaaaa gtcacaatga agacgtcaca aacgttcctg aagactaatg actcgtctaa
      721 ccccatctta ccttctgctg tttcactcaa agaaaagcca ccagctgtct ctgtgatctg
      781 ctcattctgt gttaacttgt gttctcaaca attaacaatt aaccaaaagc ctctggagct
      841 gcgtgagcat cacgatgtgt ttgtcttctg tgagcgtggc agtgtccacg aagtcaatgt
      901 gaattggttg gccaaacaca gctaatgtgg catgtgtgat tggacagcca cttaaaagtg
      961 ttaaagtgtg tttacgcaca ggattggttt tccgctgaat caggcaaaac cagcagaacc
     1021 gatctaaact cgagtataaa agtgagtagg agatgctatt cgtcccaaaa catctccacc
     1081 accatctcca ccattcctcg cctctgcttc gagaacaggt gagtgtcgag cgggatggta
     1141 aatctacaga gaatgcggag tgtagcttgt gtacatgttt ttgattaaca gagattgtat
     1201 gtgtgtgttt gtgtcagtgt gaaagggtgg gcgcgccgat cctgagaact tccgggtgag
     1261 tttggggacc cttgattgtt ctttcttttt cgctattgta aaattcatgt tatatggagg
     1321 gggcaaagtt ttcagggtgt tgtttagaat gggaagatgt cccttgtatc accatggacc
     1381 ctcatgataa ttttgtttct ttcactttct actctgttga caaccattgt ctcctcttat
     1441 tttcttttca ttttctgtaa ctttttcgtt aaactttagc ttgcatttgt aacgaatttt
     1501 taaattcact tttgtttatt tgtcagattg taagtacttt ctctaatcac ttttttttca
     1561 aggcaatcag ggtatattat attgtacttc agcacagttt tagagaacaa ttgttataat
     1621 taaatgataa ggtagaatat ttctgcatat aaattctggc tggcgtggaa atattcttat
     1681 tggtagaaac aactacaccc tggtcatcat cctgcctttc tctttatggt tacaatgata
     1741 tacactgttt gagatgagga taaaatactc tgagtccaaa ccgggcccct ctgctaacca
     1801 tgttcatgcc ttcttctctt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc
     1861 tcatcatttt ggcaaagaat tactagtgga tctcgccacc atgggttctc atcatcatca
     1921 tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga
     1981 tgacgataag gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga ataagacagg
     2041 tcacgcagtc agagctatag gtcggctgag ctcactcgag aacgtctata tcaaggccga
     2101 caagcagaag aacggcatca aggcgaactt ccacatccgc cacaacatcg aggacggcgg
     2161 cgtgcagctc gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2221 gcccgacaac cactacctga gcgtgcagtc caaactttcg aaagacccca acgagaagcg
     2281 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2341 gctgtacaag ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg
     2401 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2461 cggcgagggt gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2521 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2581 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2641 aggctacatc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2701 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2761 caaggaggac ggcaacatcc tggggcacaa gctggagtac aacctgccgg accaactgac
     2821 tgaagagcag atcgcagaat ttaaagaggc tttctcccta tttgacaagg acggggatgg
     2881 gacaataaca accaaggagc tggggacggt gatgcggtct ctggggcaga accccacaga
     2941 agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt
     3001 ccctgagttc ctgacaatga tggcaagaaa aatgaaatac agggacacgg aagaagaaat
     3061 tagagaagcg ttcggtgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct
     3121 tcgccacgtg atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat
     3181 cagggaagca gacatcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat
     3241 gacagcgaag tgagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     3301 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3361 caattcttaa ttaaggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     3421 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     3481 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3541 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3601 catcaatgta tcttaatcga gggggggccc ggtacccacc gcggtggcgg cgatctctcc
     3661 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caacccaccc
     3721 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
     3781 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
     3841 gatggctggc aactagaagg cacagtcgag gctgatcagc gggccgcttt acttgtacag
     3901 ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt
     3961 gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg cgccgggcag
     4021 ctgcacgggc ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc
     4081 cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg
     4141 ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa
     4201 gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcaggg aggagtcctg
     4261 ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc
     4321 ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt aggccttgga
     4381 gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc cacccttggt
     4441 caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat
     4501 ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt
     4561 gatgatggcc atgttatcct cctcgccctt gctcaccatg ttgtcagacc tggtaactcc
     4621 ttactgtaac ttattgcacc tgatgaacaa aagatgtgaa ctgaacgcaa gtaggaggct
     4681 aatagaggct agtacagtac aaagatcccc aaatgatgtg tgccggcctt agtggaggca
     4741 gacccctggt tatatacctg tgctgcagaa agaggggctt acaaatggat ctctctggaa
     4801 tggcattatc tcatggtggg aacagtgttg tcagcagaat cagcggggtg gactctgacc
     4861 tgagaatgac agtctagcac tgaatggctc agacaatact cttgtggcca actatcaccg
     4921 gaaaataagt gcctgccaaa acaaaccaga ccagatgaag ctacatggct gtagaacgtc
     4981 agtatttaat gtgttgtggc cacttggttc accagattta agagctaatt tttttaaata
     5041 tcacagataa tcaatcgctt tttttttttc ttgactgaaa agcttagact ggaaaattct
     5101 ttgaaaaagt ttgcactaca gaaaataaat acataacatc ctgcactgaa tgcacactat
     5161 taatggtacc cagcttttgt tccctttagt gagggttaat tgcgcgctag ggataacagg
     5221 gtaatacgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     5281 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     5341 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
     5401 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5461 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5521 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5581 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5641 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5701 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5761 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5821 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     5881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5941 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6001 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6061 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6121 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6181 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6241 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6301 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6361 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6421 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6481 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6541 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6601 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6661 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6721 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6781 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6841 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6901 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6961 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7021 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7081 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7141 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7201 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7261 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7321 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7381 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7441 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7501 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7561 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7621 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7681 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7741 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     7801 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     7861 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     7921 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     7981 cacaggaaac agctatgacc atgattacgc caagcgcgtt agggataaca gggtaatgcg
     8041 cgcg
//
