LOCUS       sequence-255702-        5965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Insulin-GLuc from the rat insulin promoter. Gaussia Luc is
            inserted within the C-peptide and is co-secreted with insulin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5965)
  AUTHORS   Kalwat MA, Wichaidit C, Nava Garcia AY, McCoy MK, McGlynn K, Hwang 
            IH, MacMillan JB, Posner BA, Cobb MH
  TITLE     Insulin promoter-driven Gaussia luciferase-based insulin secretion 
            biosensor assay for discovery of beta-cell glucose-sensing pathways.
  JOURNAL   ACS Sens. 2016 Oct 28;1(10):1208-1212. Epub 2016 Oct 12.
  PUBMED    27819058
REFERENCE   2  (bases 1 to 5965)
  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: "ACS Sens.";
            date: "2016-10-28"; volume: "1(10)"; pages: "1208-1212. Epub 2016 
            Oct 12"
COMMENT     Sequence Label: pcDNA3.1+(rInsp)-Ins-GLuc
FEATURES             Location/Qualifiers
     source          1..5965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     458..529
                     /codon_start=1
                     /product="signal sequence of human preproinsulin"
                     /label=insulin signal sequence
                     /translation="MALWMRLLPLLALLALWGPDPAAA"
     primer_bind     complement(1344..1361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1350..1574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1620..2048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1707..1726)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1917..1938
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2057..2077)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2062..2391
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2242..2377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2304..2323
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2458..3252
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2512..2531)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3122..3141
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3426..3547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3463..3482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3517..3536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3596..3612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3596..3612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3609..3631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3620..3636
                     /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(3644..3650,3651..3668,3669..3674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3689..3710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3827..3844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3998..4586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4078..4097)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4757..5548,5549..5617))
                     /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     5380..5399
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5618..5722)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5797..5816)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtacgggcca gatatacgcg tgctgagcta agaatccagc tatcaataga aactatgaaa
       61 cagttccagg gacaaagata ccaggtcccc aacaactgca actttctggg aaatgaggtg
      121 gaaaatgctc agccaaggaa aaagagggcc ttaccctctc tgggacaatg attgtgctgt
      181 gaactgcttc atcaggccat ctggcccctt gttaataatc taattaccct aggtctaagt
      241 agagttgttg acgtccaatg agcgctttct gcagacttag cactaggcaa gtgtttggaa
      301 attacagctt cagcccctct cgccatctgc ctacctaccc ctcctagagc ccttaatggg
      361 ccaaacggca aagtccaggg ggcagagagg aggtgctttg gactataaag ctagtggaga
      421 cccagtaact cccaagctag cgtttaaact taagcttatg gccctgtgga tgcgcctcct
      481 gcccctgctg gcgctgctgg ccctctgggg acctgaccca gccgcagcct ttgtgaacca
      541 acacctgtgc ggctcacacc tggtggaagc tctctaccta gtgtgcgggg aacgaggctt
      601 cttctacaca cccaagaccc gccgggaggc agaggacctg caggtggggc aggtggagct
      661 gggcgggggc cctggatcca agcccaccga gaacaacgaa gacttcaaca tcgtggccgt
      721 ggccagcaac ttcgcgacca cggatctcga tgctgaccgc gggaagttgc ccggcaagaa
      781 gctgccgctg gaggtgctca aagagatcga agccaatgcc cggaaagctg gctgcaccag
      841 gggctgtctg atctgcctgt cccacatcaa gtgcacgccc aagatgaaga agttcatccc
      901 aggacgctgc cacacctacg aaggcgacaa agagtccgca cagggcggca taggcgaggc
      961 gatcgtcgac attcctgaga ttcctgggtt caaggacttg gagcccatcg agcagttcat
     1021 cgcacaggtc gatctgtgtg tggactgcac aactggctgc ctcaaagggc ttgccaacgt
     1081 gcagtgttct gacctgctca agaagtggct gccgcaacgc tgtgcgacct ttgccagcaa
     1141 gatccagggc caggtggaca agatcaaggg ggccggtggt gacgaattcg gtgcaggcag
     1201 cctgcagccc ttggccctgg aggggtccct gcagaagcgt ggcattgtgg aacaatgctg
     1261 taccagcatc tgctccctct accagctgga gaactactgc aactagctcg agtctagagg
     1321 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1381 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1441 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1501 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1561 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1621 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1681 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1741 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1801 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1861 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1921 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1981 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2041 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2101 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2161 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2221 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2281 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     2341 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     2401 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     2461 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     2521 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     2581 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     2641 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     2701 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     2761 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     2821 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     2881 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     2941 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3001 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3061 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3121 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3181 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3241 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     3301 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     3361 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     3421 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3481 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3541 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     3601 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3661 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3721 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3781 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3841 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3901 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3961 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4021 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4081 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4141 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4201 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4261 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4321 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4381 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4441 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4501 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4561 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4621 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4681 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4741 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4801 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4861 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4921 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4981 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5041 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5101 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5161 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5221 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5281 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5341 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5401 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5461 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5521 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5581 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5641 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5701 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     5761 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     5821 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     5881 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     5941 taggcgtttt gcgctgcttc gcgat
//
