LOCUS       sequence-350664-        5993 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial vector for expression of an N-terminal GST fusion of 
            PolB(K206A/K244A/L301R/V303R/V306R) with a TEV protease site located
            between the GST tag and PolB(K206A/K244A/L301R/V303R/V306R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5993)
  AUTHORS   Fang Q, Andrews J, Sharma N, Wilk A, Clark J, Slyskova J, Koczor CA,
            Lans H, Prakash A, Sobol RW
  TITLE     Stability and sub-cellular localization of DNA polymerase beta is 
            regulated by interactions with NQO1 and XRCC1 in response to 
            oxidative stress.
  JOURNAL   Nucleic Acids Res. 2019 Jul 9;47(12):6269-6286. doi: 
            10.1093/nar/gkz293.
  PUBMED    31287140
REFERENCE   2  (bases 1 to 5993)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2019-07-9- 9"; volume: "47"; issue: 
            "12"; pages: "6269-6286"
COMMENT     Sequence Label: pGEX4T3-GST-PolB(K206A/K244A/L301R/V303R/V306R)
FEATURES             Location/Qualifiers
     source          1..5993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(531..536,537..554,555..561)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    569..585
                     /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     593..609
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     593..609
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             605..1132
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(625..642)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(625..641)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(634..656)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1234..1239,1240..1255,1256..1262)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1270..1286
                     /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)."
     CDS             1309..1962
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1920..1942
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1969..1986
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1987..2007
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3094..3116)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3261..3279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3347..3451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3452..3520,3521..4312)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3670..3689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4483..5071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4972..4991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5225..5242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5315..5392
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5393..5993,1..482)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5412..5431)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccccaggc tttacacttt
      541 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      601 agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg actgggaaaa
      661 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
      721 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
      781 gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct
      841 tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc
      901 catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa
      961 tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca
     1021 gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt gcaccaatgc
     1081 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     1141 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     1201 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
     1261 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat gtcccctata
     1321 ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt
     1381 gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa
     1441 aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa
     1501 ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
     1561 tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac
     1621 ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc
     1681 aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat
     1741 ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac
     1801 atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
     1861 gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag
     1921 ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct ggttccgcgt
     1981 ggatccgaaa acctgtattt tcagggctcg aattcccggg tcgacatgag caaacggaag
     2041 gcgccgcagg agactctcaa cgggggaatc accgacatgc tcacagaact cgcaaacttt
     2101 gagaagaacg tgagccaagc tatccacaag tacaatgctt acagaaaagc agcatctgtt
     2161 atagcaaaat acccacacaa aataaagagt ggagctgaag ctaagaaatt gcctggagta
     2221 ggaacaaaaa ttgctgaaaa gattgatgag tttttagcaa ctggaaaatt acgtaaactg
     2281 gaaaagattc ggcaggatga tacgagttca tccatcaatt tcctgactcg agttagtggc
     2341 attggtccat ctgctgcaag gaagtttgta gatgaaggaa ttaaaacact agaagatctc
     2401 agaaaaaatg aagataaatt gaaccatcat cagcgaattg ggctgaaata ttttggggac
     2461 tttgaaaaaa gaattcctcg tgaagagatg ttacaaatgc aagatattgt actaaatgaa
     2521 gttaaaaaag tggattctga atacattgct acagtctgtg gcagtttcag aagaggtgca
     2581 gagtccagtg gtgacatgga tgttctcctg acccatccca gcttcacttc agaatcaacc
     2641 gcacagccaa aactgttaca tcaggttgtg gagcagttac aaaaggttca ttttatcaca
     2701 gataccctgt caaagggtga gacaaagttc atgggtgttt gccagcttcc cagtgcaaat
     2761 gatgaaaaag aatatccaca cagaagaatt gatatcaggt tgatacccaa agatcagtat
     2821 tactgtggtg ttctctattt cactgggagt gatattttca ataagaatat gagggctcat
     2881 gccctagaaa agggtttcac aatcaatgag tacaccatcc gtccccgggg acgcactgga
     2941 cgtgcaggag aacccctgcc agtggatagt gaaaaagaca tctttgatta catccagtgg
     3001 aaataccggg aacccaagga ccggagcgaa tgagcggccg catcgtgact gactgacgat
     3061 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3121 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3181 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3241 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3301 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3361 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3421 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3481 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3541 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3601 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3661 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     3721 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3781 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3841 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3901 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3961 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     4021 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4081 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4141 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4201 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4261 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4321 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4381 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4441 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4501 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4561 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4621 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4681 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4741 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4801 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4861 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4921 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4981 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5041 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5101 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5161 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5221 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5281 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt
     5341 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     5401 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     5461 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     5521 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     5581 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     5641 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     5701 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     5761 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     5821 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     5881 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     5941 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gct
//
