LOCUS       sequence-174328-        6209 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 6209)
  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 6209)
  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..6209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     778..797
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        778..796
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     832..903
                     /codon_start=1
                     /product="signal sequence of human preproinsulin"
                     /label=insulin signal sequence
                     /translation="MALWMRLLPLLALLALWGPDPAAA"
     primer_bind     complement(1718..1735)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1724..1948
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1994..2422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2081..2100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2291..2312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2431..2451)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2436..2765
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2616..2751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2678..2697
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2832..3626
                     /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(2886..2905)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3496..3515
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3800..3921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3837..3856)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3891..3910
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3970..3986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3970..3986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3983..4005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3994..4010
                     /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(4018..4024,4025..4042,4043..4048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4063..4084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4201..4218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4372..4957)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4452..4471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5128..5919,5920..5988))
                     /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     5751..5770
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5989..6093)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6168..6187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
       61 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      121 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      781 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tatggccctg
      841 tggatgcgcc tcctgcccct gctggcgctg ctggccctct ggggacctga cccagccgca
      901 gcctttgtga accaacacct gtgcggctca cacctggtgg aagctctcta cctagtgtgc
      961 ggggaacgag gcttcttcta cacacccaag acccgccggg aggcagagga cctgcaggtg
     1021 gggcaggtgg agctgggcgg gggccctgga tccaagccca ccgagaacaa cgaagacttc
     1081 aacatcgtgg ccgtggccag caacttcgcg accacggatc tcgatgctga ccgcgggaag
     1141 ttgcccggca agaagctgcc gctggaggtg ctcaaagaga tcgaagccaa tgcccggaaa
     1201 gctggctgca ccaggggctg tctgatctgc ctgtcccaca tcaagtgcac gcccaagatg
     1261 aagaagttca tcccaggacg ctgccacacc tacgaaggcg acaaagagtc cgcacagggc
     1321 ggcataggcg aggcgatcgt cgacattcct gagattcctg ggttcaagga cttggagccc
     1381 atcgagcagt tcatcgcaca ggtcgatctg tgtgtggact gcacaactgg ctgcctcaaa
     1441 gggcttgcca acgtgcagtg ttctgacctg ctcaagaagt ggctgccgca acgctgtgcg
     1501 acctttgcca gcaagatcca gggccaggtg gacaagatca agggggccgg tggtgacgaa
     1561 ttcggtgcag gcagcctgca gcccttggcc ctggaggggt ccctgcagaa gcgtggcatt
     1621 gtggaacaat gctgtaccag catctgctcc ctctaccagc tggagaacta ctgcaactag
     1681 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     1741 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1801 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1861 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1921 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     1981 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2041 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2101 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2161 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2221 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2281 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2341 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2401 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2461 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2521 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2581 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2641 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     2701 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2761 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     2821 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     2881 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     2941 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3001 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3061 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3121 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3181 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3241 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3301 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3361 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3421 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3481 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3541 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3601 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     3661 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3721 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3781 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3841 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3901 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3961 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4021 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4081 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4141 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4201 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4261 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4321 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4381 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4441 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4501 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4561 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4621 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4681 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4741 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4801 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4861 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg
     4921 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4981 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5041 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5101 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5161 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5221 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5281 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5341 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5401 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5461 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5521 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5581 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5641 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5701 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5761 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5821 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5881 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5941 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6001 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6061 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6121 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     6181 gatgccgcat agttaagcca gtatctgct
//
