LOCUS       sequence-84089-e        4785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  842 bp insert from mouse Ins2 gene used as a control for 
            methylation-specific PCR assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4785)
  AUTHORS   Husseiny MI, Kuroda A, Kaye AN, Nair I, Kandeel F, Ferreri K
  TITLE     Development of a quantitative methylation-specific polymerase chain 
            reaction method for monitoring beta cell death in type 1 diabetes.
  JOURNAL   PLoS One. 2012;7(10):e47942. doi: 10.1371/journal.pone.0047942. Epub
            2012 Oct 29.
  PUBMED    23144715
REFERENCE   2  (bases 1 to 4785)
  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: "PLoS One.";
            date: "2012"; volume: "7(10):e47942. doi"; pages: " 
            10.1371/journal.pone.0047942. Epub 2012 Oct 29"
COMMENT     Sequence Label: pCR2.1-Ins2-842
FEATURES             Location/Qualifiers
     source          1..4785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1218..1237)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1219..1237)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1244..1261)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1244..1260)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1253..1275)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1401..1829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1698..1719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2173..2967
                     /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(2227..2246)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2837..2856
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2985..3053,3054..3845)
                     /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(3203..3222)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4016..4604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4505..4524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4758..4775
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttgagctc
      301 ggaccattaa gtgccttgct gcctgaattc tgctttcctt ctacctctga gagagagctg
      361 gggactcggc tgagttaaga acccagctat caattggaac tgtgaaacag tccaagggac
      421 aaagatacta ggtccccaac tgcaacttcc tggggaatga tgtggaaaaa tgctcagcca
      481 aggacaaaga aagcatcacc cactctggaa caatgtcccc tgctgtgaac tggttcatca
      541 ggccatcagg gccccttgtt aagactctaa ttaccctagg actaagtaga ggtgttgacg
      601 tccaatgagc gctttctgca gacctagcac cagggaagtg tttggaaact gcagcttcag
      661 cccctctggc catctgctga cctaccccac ctggagccct taatgggtca aacagcaaag
      721 tccagggggc agagaggagg tgctttggtc tataaaggta gtggggaccc agtaaccacc
      781 agccctaagt gatccgctac aatcaaaaac catcagcaag caggaaggta ctcttctcag
      841 tgggcctggc tccccagcta agacctcagg gacttgaggt aggatatagc ctcctctctt
      901 acgtgaaact tttgctatcc tcaacccagc ctatcttcca ggttattgtt tcaacatggc
      961 cctgtggatg cgcttcctgc ccctgctggc cctgctcttc ctctgggagt cccaccccac
     1021 ccaggctttt gtcaagcagc acctttgtgg ttcccacctg gtggaggctc tctacctggt
     1081 gtgtggggag cgtggcttct tctacacacc catgtcccgc cgtgaagtgg aggacccaca
     1141 agccatggaa gggcgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat
     1201 ctagagggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac
     1261 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     1321 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     1381 gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1441 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1501 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1561 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1621 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1681 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1741 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1801 gctgatttaa caaaaattta acgcgaattt taacaaaatt cagggcgcaa gggctgctaa
     1861 aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     1921 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     1981 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     2041 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     2101 ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga
     2161 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2221 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2281 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2341 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2401 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2461 gtgccggggc aggatctcct gtcatcccac cttgctcctg ccgagaaagt atccatcatg
     2521 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2581 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2641 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2701 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2761 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2821 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2881 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2941 tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt attcaacatt
     3001 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3061 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3121 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3181 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3241 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3301 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3361 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3421 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3481 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3541 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3601 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3661 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3721 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3781 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3841 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3901 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3961 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4021 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4081 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4141 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4201 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4261 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4321 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4381 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4441 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4501 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4561 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4621 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4681 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4741 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
