LOCUS       sequence-255704-        6212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Insulin-GLuc from the CMV 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 6212)
  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 6212)
  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+CMV-Ins-GLuc
FEATURES             Location/Qualifiers
     source          1..6212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     683..754
                     /codon_start=1
                     /product="signal sequence of human preproinsulin"
                     /label=insulin signal sequence
                     /translation="MALWMRLLPLLALLALWGPDPAAA"
     primer_bind     complement(1569..1586)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1575..1799
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1845..2273
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1932..1951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2142..2163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2282..2302)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2287..2616
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2467..2602
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2529..2548
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2683..3477
                     /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(2737..2756)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3347..3366
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3651..3772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3688..3707)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3742..3761
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3821..3837)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3821..3837)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3834..3856)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3845..3861
                     /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(3869..3875,3876..3893,3894..3899))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3914..3935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4052..4069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4223..4811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4303..4322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4982..5773,5774..5842))
                     /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     5605..5624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5843..5947)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6022..6041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttatggccct gtggatgcgc ctcctgcccc tgctggcgct
      721 gctggccctc tggggacctg acccagccgc agcctttgtg aaccaacacc tgtgcggctc
      781 acacctggtg gaagctctct acctagtgtg cggggaacga ggcttcttct acacacccaa
      841 gacccgccgg gaggcagagg acctgcaggt ggggcaggtg gagctgggcg ggggccctgg
      901 atccaagccc accgagaaca acgaagactt caacatcgtg gccgtggcca gcaacttcgc
      961 gaccacggat ctcgatgctg accgcgggaa gttgcccggc aagaagctgc cgctggaggt
     1021 gctcaaagag atcgaagcca atgcccggaa agctggctgc accaggggct gtctgatctg
     1081 cctgtcccac atcaagtgca cgcccaagat gaagaagttc atcccaggac gctgccacac
     1141 ctacgaaggc gacaaagagt ccgcacaggg cggcataggc gaggcgatcg tcgacattcc
     1201 tgagattcct gggttcaagg acttggagcc catcgagcag ttcatcgcac aggtcgatct
     1261 gtgtgtggac tgcacaactg gctgcctcaa agggcttgcc aacgtgcagt gttctgacct
     1321 gctcaagaag tggctgccgc aacgctgtgc gacctttgcc agcaagatcc agggccaggt
     1381 ggacaagatc aagggggccg gtggtgacga attcggtgca ggcagcctgc agcccttggc
     1441 cctggagggg tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc
     1501 cctctaccag ctggagaact actgcaacta gctcgagtct agagggcccg tttaaacccg
     1561 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1621 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1681 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1741 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     1801 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     1861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2041 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2161 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2281 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2341 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2401 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2461 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2521 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     2581 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     2641 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2701 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2761 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2821 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     2881 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2941 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3001 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3061 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3121 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3181 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     3241 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3301 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3361 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3421 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3481 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     3541 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3601 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3661 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3721 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3781 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3841 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3901 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3961 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4021 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4081 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4141 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4201 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4261 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4321 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4381 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4441 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4501 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4561 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4621 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4681 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4741 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4801 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4861 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4921 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4981 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5041 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5101 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5161 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5221 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5281 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5341 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5401 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5461 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5521 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5581 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5641 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5701 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5761 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5821 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5881 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5941 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6001 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     6061 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     6121 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     6181 gcttcgcgat gtacgggcca gatatacgcg tt
//
