LOCUS       sequence-267648-        8069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of N-terminally FLAG-tagged DNA polymerase iota
            containing mutations K440A, K444A and K446A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8069)
  AUTHORS   Ashton NW, Valles GJ, Jaiswal N, Bezsonova I, Woodgate R
  TITLE     DNA Polymerase iota Interacts with Both the TRAF-like and UBL1-2 
            Domains of USP7.
  JOURNAL   J Mol Biol. 2021 Jan 22;433(2):166733. doi: 
            10.1016/j.jmb.2020.166733. Epub 2020 Dec 3.
  PUBMED    33279577
REFERENCE   2  (bases 1 to 8069)
  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: "J Mol 
            Biol."; date: "2021-01-22"; volume: "433(2):166733. doi"; pages: " 
            10.1016/j.jmb.2020.166733. Epub 2020 Dec 3"
COMMENT     Sequence Label: pCMV6-AN-DDK_3KA POLI
FEATURES             Location/Qualifiers
     source          1..8069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     165..182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1046
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3274..3290)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3301..3923
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3500..3519)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3947..3967)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3952..4281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4132..4267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4348..5142
                     /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(4402..4421)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5012..5031
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5316..5437
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5353..5372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5407..5426
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5486..5502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5499..5521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5510..5526
                     /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(5534..5540,5541..5558,5559..5564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5579..5600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5717..5734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5888..6476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5968..5987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6647..7438,7439..7507))
                     /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     7270..7289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7508..7612)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7639..8067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7726..7745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7936..7957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga tctgccacca
     1021 tggattacaa ggatgacgac gataaggcga tcgccgatat ccatatggag ctggccgacg
     1081 tgggagccgc tgccagctcc cagggggtgc acgatcaggt gctgccaact cccaacgctt
     1141 ctagtagagt gatcgtgcat gtggacctgg attgctttta cgcccaggtg gagatgatta
     1201 gcaatcctga actgaaggac aaaccactgg gcgtgcagca gaaatacctg gtggtgacat
     1261 gcaactatga agctcgcaag ctgggggtga agaaactgat gaatgtgcgg gacgccaagg
     1321 agaaatgtcc ccagctggtg ctggtgaacg gggaggatct gacccggtac agagaaatgt
     1381 cttataaggt gacagagctg ctggaggagt tcagtcctgt ggtggagcgc ctgggcttcg
     1441 acgaaaattt tgtggatctg acagagatgg tggaaaagcg gctgcagcag ctgcagtctg
     1501 acgagctgag tgccgtgact gtgagtggac acgtgtacaa caatcagtca attaacctgc
     1561 tggatgtgct gcatatcaga ctgctggtgg gcagccagat tgctgccgag atgagggaag
     1621 ctatgtataa ccagctggga ctgaccggat gtgctggagt ggcctcaaat aagctgctgg
     1681 ccaaactggt cagcggcgtg tttaaaccca accagcagac agtgctgctg cctgagtctt
     1741 gccagcacct gatccatagt ctgaatcaca tcaaggaaat tcccggcatc gggtacaaga
     1801 ctgctaaatg tctggaggcc ctgggaatca acagcgtgcg cgacctgcag accttttccc
     1861 ctaagattct ggagaaagaa ctgggcattt ccgtggccca gagaatccag aagctgtctt
     1921 tcggagagga caattcccca gtgatcctgt ctggcccccc tcagagcttc tccgaggaag
     1981 atagctttaa gaaatgctca agcgaggtgg aagctaagaa caaaattgag gaactgctgg
     2041 cctccctgct gaatagggtg tgtcaggatg ggcgcaaacc tcacaccgtg cggctgatca
     2101 ttcggagata ctcctctgag aagcactatg gaagggaatc acgccagtgc cctattccaa
     2161 gccatgtgat ccagaagctg ggaacaggca actatgacgt gatgactcct atggtggaca
     2221 tcctgatgaa actgttccgc aacatggtga atgtgaagat gcctttccat ctgaccctgc
     2281 tgtctgtgtg cttttgtaac ctgaaagctc tgaatacagc caagaaaggg ctgattgact
     2341 actatctgat gccatctctg agtaccacat cacggagcgg cgcccactcc ttcgccatgg
     2401 ccgacactca catggaagac tttcctaagg ataaagagac caatagagat ttcctgccaa
     2461 gtggcaggat cgagtcaacc cggacaagag aatctcccct ggacactacc aacttcagta
     2521 aggagaagga tattaacgaa tttccactgt gcagcctgcc tgagggagtg gaccaggaag
     2581 tgtttaagca gctgcctgtg gatattcagg aggaaatcct gagtgggaag tcaagagaga
     2641 aattccaggg gaagggcagc gtgtcttgtc cactgcacgc ttccaggggc gtgctgagct
     2701 tcttttccaa gaaacagatg caggacatcc ccattaaccc tagagatcat ctgagttcaa
     2761 gcaaacaggt gtcctctgtg tccccatgtg agcccggcac ctctgggttt aacagttcaa
     2821 gctcctctta catgagttca cagaaggact attcctacta tctggacaat cggctgaaag
     2881 atgaaagaat ctctcaggga cctaaggagc cacagggctt ccactttaca aacagcaatc
     2941 cagctgtgtc agccttccat agctttccca acctgcagag cgagcagctg ttctccagaa
     3001 atcacacaac tgacagtcat aaacagactg tggccaccga ttcacacgaa ggcctgacag
     3061 agaataggga accagacagc gtggatgaga agattacttt cccatccgac atcgatcccc
     3121 aggtgtttta cgagctgccc gaagctgtgc agaaagagct gctggccgaa tggaagagga
     3181 ccgggtccga tttccacatc ggacataagt aaggatccaa gcttctcgag acgcgttaag
     3241 cggccgcact cgaggtttaa acggccggcc gcggtcatag ctgtttcctg aacagatccc
     3301 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     3361 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     3421 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     3481 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     3541 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     3601 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     3661 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     3721 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     3781 attttaaaat aactatacca gcaggaggac gtccagacac agcataggct acctggccat
     3841 gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg cgttgggtcc
     3901 actcagtaga tgcctgttga attgggtacg cggccagctt ggctgtggaa tgtgtgtcag
     3961 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4021 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4081 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4141 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4201 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4261 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4321 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4381 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4441 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4501 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4561 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4621 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4681 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4741 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4801 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4861 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4921 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4981 ctgggtgtgg ccgaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5041 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5101 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5161 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5221 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5281 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5341 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5401 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5461 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5521 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5581 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5641 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5701 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5761 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5821 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5881 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5941 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6001 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6061 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6121 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6181 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6241 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6301 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6361 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6421 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6481 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6541 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6601 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6661 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6721 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6781 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6841 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6901 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6961 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7021 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7081 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7141 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7201 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7261 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7321 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7381 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7441 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7501 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7561 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7621 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     7681 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     7741 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     7801 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     7861 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     7921 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     7981 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     8041 tgatttaaca aaaatttaac gcgaatttt
//
