LOCUS       sequence-387969-        6744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a human PTPN22 mutant (intron 18-runon) with
            FLAG tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6744)
  AUTHORS   Ge Y, Onengut-Gumuscu S, Quinlan AR, Mackey AJ, Wright JA, Buckner 
            JH, Habib T, Rich SS, Concannon P
  TITLE     Targeted Deep Sequencing in Multiple-Affected Sibships of European 
            Ancestry Identifies Rare Deleterious Variants in PTPN22 That Confer 
            Risk for Type 1 Diabetes.
  JOURNAL   Diabetes. 2016 Mar;65(3):794-802. doi: 10.2337/db15-0322. Epub 2015 
            Dec 2.
  PUBMED    26631741
REFERENCE   2  (bases 1 to 6744)
  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"; doi: "10.2337/db15-0322";
            journalName: "Diabetes"; date: "2016-03"; volume: "65"; issue: "3"; 
            pages: "794-802"
COMMENT     Sequence Label: FLAG-In18runon-PTPN22
FEATURES             Location/Qualifiers
     source          1..6744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        62..365
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        366..569
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     520..540
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        615..633
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             677..700
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3240..3259)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3241..3259)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3533..3654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3570..3589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3624..3643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3661..4116)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3798..3819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4010..4029
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4143..4247
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4249..4606
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4249..4440
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4272..4292)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4457..4592
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4641..5435
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4641..5435
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4695..4714)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5305..5324
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5623..5642)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5667..5714
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6043..6631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6532..6551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
       61 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      121 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      181 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      241 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      301 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      361 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      421 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      481 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      541 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgat
      601 tacgccaagc tcgaaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt
      661 ggcggccgcc accatggatt acaaggatga cgacgataag agcccgggcg gatccatgga
      721 ccaaagagaa attctgcaga agttcctgga tgaggcccaa agcaagaaaa ttactaaaga
      781 ggagtttgcc aatgaatttc tgaagctgaa aaggcaatct accaagtaca aggcagacaa
      841 aacctatcct acaactgtgg ctgagaagcc caagaatatc aagaaaaaca gatataagga
      901 tattttgccc tatgattata gccgggtaga actatccctg ataacctctg atgaggattc
      961 cagctacatc aatgccaact tcattaaggg agtttatgga cccaaggctt atattgccac
     1021 ccagggtcct ttatctacaa ccctcctgga cttctggagg atgatttggg aatatagtgt
     1081 ccttatcatt gttatggcat gcatggagta tgaaatggga aagaaaaagt gtgagcgcta
     1141 ctgggctgag ccaggagaga tgcagctgga atttggccct ttctctgtat cctgtgaagc
     1201 tgaaaaaagg aaatctgatt atataatcag gactctaaaa gttaagttca atagtgaaac
     1261 tcgaactatc taccagtttc attacaagaa ttggccagac catgatgtac cttcatctat
     1321 agaccctatt cttgagctca tctgggatgt acgttgttac caagaggatg acagtgttcc
     1381 catatgcatt cactgcagtg ctggctgtgg aaggactggt gttatttgtg ctattgatta
     1441 tacatggatg ttgctaaaag atgggataat tcctgagaac ttcagtgttt tcagtttgat
     1501 ccgggaaatg cggacacaga ggccttcatt agttcaaacg caggaacaat atgaactggt
     1561 ctacaatgct gtattagaac tatttaagag acagatggat gttatcagag ataaacattc
     1621 tggaacagag agtcaagcaa agcattgtat tcctgagaaa aatcacactc tccaagcaga
     1681 ctcttattct cctaatttac caaaaagtac cacaaaagca gcaaaaatga tgaaccaaca
     1741 aaggacaaaa atggaaatca aagaatcttc ttcctttgac tttaggactt ctgaaataag
     1801 tgcaaaagaa gagctagttt tgcaccctgc taaatcaagc acttcttttg actttctgga
     1861 gctaaattac agttttgaca aaaatgctga cacaaccatg aaatggcaga caaaggcatt
     1921 tccaatagtt ggggagcctc ttcagaagca tcaaagtttg gatttgggct ctcttttgtt
     1981 tgagggatgt tctaattcta aacctgtaaa tgcagcagga agatatttta attcaaaggt
     2041 gccaataaca cggaccaaat caactccttt tgaattgata cagcagagag aaaccaagga
     2101 ggtggacagc aaggaaaact tttcttattt ggaatctcaa ccacatgatt cttgttttgt
     2161 agagatgcag gctcaaaaag taatgcatgt ttcttcagca gaactgaatt attcactgcc
     2221 atatgactct aaacaccaaa tacgtaatgc ctctaatgta aagcaccatg actctagtgc
     2281 tcttggtgta tattcttaca tacctttagt ggaaaatcct tatttttcat catggcctcc
     2341 aagtggtacc agttctaaga tgtctcttga tttacctgag aagcaagatg gaactgtttt
     2401 tccttcttct ctgttgccaa catcctctac atccctcttc tcttattaca attcacatga
     2461 ttctttatca ctgaattctc caaccaatat ttcctcacta ttgaaccagg agtcagctgt
     2521 actagcaact gctccaagga tagatgatga aatcccccct ccacttcctg tacggacacc
     2581 tgaatcattt attgtggttg aggaagctgg agaattctca ccaaatgttc ccaaatcctt
     2641 atcctcagct gtgaaggtaa aaattggaac atcactggaa tggggtggaa catctgaacc
     2701 aaagaaattt gatgactctg tgatacttag accaagcaag agtgtaaaac tccgaagtcc
     2761 taaatcagaa ctacatcaag atcgttcttc tcccccacct cctctcccag aaagaactct
     2821 agagtccttc tttcttgccg atgaagattg tatgcaggcc caatctatag aaacatattc
     2881 tactagctat cctgacacca tggaaaattc aacatcttca aaacagacac tgaagactcc
     2941 tggaaaaagt ttcacaagga gtaacgtaaa gaagataaga gttttgggaa acatgccgga
     3001 aactttccct tactgactcg agggggggcc cggtacctta attaattaag gtaccaggta
     3061 agtgtaccca attcgcccta tagwrarymg wmwctgaaga ctcctggaaa aagtttcaca
     3121 aggagtaacg taaagaagat aagagttttg ggaaacatgc cggaaacttt cccttactga
     3181 ctcgaggggg ggcccggtac cttaattaat taaggtacca ggtaagtgta cccaattcgc
     3241 cctatagtga gtcgtattac aattcactcg atcgcccttc ccaacagttg cgcagcctga
     3301 atggcgaatg gagatccaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt
     3361 ttgtgtattt tagattcaca gtcccaaggc tcatttcagg cccctcagtc ctcacagtct
     3421 gttcatgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     3481 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     3541 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3601 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt
     3661 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3721 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3781 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3841 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3901 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3961 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4021 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4081 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg
     4141 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4201 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatcct gaggcggaaa
     4261 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4321 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4381 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4441 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4501 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     4561 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga
     4621 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4681 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4741 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4801 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     4861 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4921 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4981 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     5041 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     5101 atcaggatga tctggacgaa gaacatcagg ggctcgcgcc agccgaactg ttcgccaggc
     5161 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     5221 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     5281 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gaacttggcg
     5341 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5401 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     5461 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5521 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5581 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac
     5641 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt
     5701 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     5761 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     5821 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     5881 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5941 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6001 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6061 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6121 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6181 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6241 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6301 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6361 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6421 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6481 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6541 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6601 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6661 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6721 ggataaccgt attaccgcca tgca
//
