LOCUS       sequence-395334-        6750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy vector for expressing the catalytic domain (amino acids 
            1-419) of human DNA polymerase iota in E. coli with an N-terminal 6x
            His tag. C-terminal truncation mutant of pJRM919_WT His POLI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6750)
  AUTHORS   Ashton NW, Jaiswal N, Cestari Moreno N, Semenova IV, D'Orlando DA, 
            Teatin Latancia M, McIntyre J, Woodgate R, Bezsonova I
  TITLE     A novel interaction between RAD23A/B and Y-family DNA polymerases.
  JOURNAL   J Mol Biol. 2023 Nov 5:168353. doi: 10.1016/j.jmb.2023.168353.
  PUBMED    37935254
REFERENCE   2  (bases 1 to 6750)
  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.
            2023 Nov 5:168353. doi: 10.1016/j.jmb.2023.168353."
COMMENT     Sequence Label: pJRM919_WT aa 1-419 His POLI
FEATURES             Location/Qualifiers
     source          1..6750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             17..39
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             58..75
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             85..108
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1417..1434
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1487..1505)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1501..1548
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1585..1943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1672..1691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1882..1903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1958..1974)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1958..1974)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1982..1998
                     /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(2006..2012,2013..2030,2031..2036))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2051..2072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(2600..3550)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTKYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3598..3820
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(4243..5058)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4243..5058)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4966..4985
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5050..5069
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        5467..5544
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5545..6627
                     /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(5564..5583)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6692..6708
                     /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)."
     protein_bind    6725..6741
                     /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)."
ORIGIN
        1 gaattctaga aataattttg tttaacttta agaaggagat ataacatggg cagcagccat
       61 catcatcatc atcacagcag cggcctggaa gttctgttcc aggggcccca tatggaactg
      121 gcggatgttg gcgccgcggc gagcagccag ggcgttcatg atcaggtgtt accaaccccg
      181 aacgcgagca gccgtgtgat tgtgcatgtg gatctggatt gcttttatgc gcaggtggaa
      241 atgattagca acccggaact gaaagataaa ccgctgggcg tgcagcagaa atatctggtg
      301 gtgacctgca actacgaggc ccgtaaactg ggcgtgaaaa aactgatgaa cgtgcgtgat
      361 gcgaaagaaa aatgcccgca gctggtgctg gtgaacggcg aagatctgac ccgttatcgt
      421 gaaatgagct ataaagtgac cgaactgctg gaagaattta gcccggtggt ggaacgtctg
      481 ggctttgatg aaaactttgt ggatctgacc gaaatggtgg aaaaacgtct gcagcagctg
      541 cagagcgatg aactgagcgc ggtgaccgtg agcggccatg tgtataacaa ccagagcatt
      601 aacctgctgg atgtgctgca tattcgtctg ctggtgggca gccagattgc ggcggaaatg
      661 cgtgaagcga tgtataacca gctgggcctg accggctgcg cgggcgtggc gagcaacaaa
      721 ctgctggcga aactggtgag cggcgtgttt aaaccgaacc agcagaccgt gctgctgccg
      781 gaaagctgcc agcatctgat tcatagcctg aaccatatta aagaaattcc gggcattggc
      841 tataaaaccg cgaaatgcct ggaagcgctg ggcattaaca gcgtgcgtga tctgcagacc
      901 tttagcccga aaattctgga aaaagaactg ggcattagcg tggcgcagcg tattcagaaa
      961 ctgagctttg gcgaagataa cagcccggtg attctgagcg gtccgccaca aagctttagc
     1021 gaagaagata gctttaaaaa atgcagcagc gaagtggaag cgaaaaacaa aattgaggaa
     1081 ttactggcca gcctgctgaa ccgtgtgtgc caggatggcc gtaaaccgca taccgtgcgt
     1141 ctgattattc gtcgttatag cagcgaaaaa cattatggcc gtgaaagccg tcagtgcccg
     1201 attccgagcc atgtgattca gaaactgggc accggcaact atgatgtgat gaccccgatg
     1261 gtggatattc tgatgaaact gtttcgtaac atggtgaacg tgaaaatgcc gtttcatctg
     1321 accctgctga gcgtgtgctt ttgcaacctg aaagcgctga acaccgcgta aataaataag
     1381 tcggcctgcg aattcgtcga cgcggccgca ctcgagcacc accaccacca ccactgagat
     1441 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1501 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1561 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1621 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1681 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1741 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1801 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1861 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1921 tctcggtcta ttcttttgat ttaaattcgt aatcatggtc atagctgttt cctgtgtgaa
     1981 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2041 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2101 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2161 gtttgcgtat tgggcgctat ttcttccaga attgccatga ttttttcccc acgggaggcg
     2221 tcactggctc ccgtgttgtc ggcagctttg attcgataag cagcatcgcc tgtttcaggc
     2281 tgtctatgtg tgactgttga gctgtaacaa gttgtctcag gtgttcaatt tcatgttcta
     2341 gttgctttgt tttactggtt tcacctgttc tattaggtgt tacatgctgt tcatctgtta
     2401 cattgtcgat ctgttcatgg tgaacagctt taaatgcacc aaaaactcgt aaaagctctg
     2461 atgtatctat cttttttaca ccgttttcat ctgtgcatat atggacagtt ttccctttga
     2521 tatgtaacgg tgaacagttg ttctactttt gtttgttagt cttgatgctt cactgataga
     2581 tacaagagcc ataagaacct cagatccttc cgtatttagc cagtatgttc tctagtgtgg
     2641 ttcgttgttt ttgcgtgagc catgagaacg aaccattgag atcatactta ctttgcatgt
     2701 cactcaaaaa ttttgcctca aaactggtga gctgaatttt tgcagttaaa gcatcgtgta
     2761 gtgtttttct tagtccgtta tgtaggtagg aatctgatgt aatggttgtt ggtattttgt
     2821 caccattcat ttttatctgg ttgttctcaa gttcggttac gagatccatt tgtctatcta
     2881 gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg cttatcaacc accaatttca
     2941 tattgctgta agtgtttaaa tctttactta ttggtttcaa aacccattgg ttaagccttt
     3001 taaactcatg gtagttattt tcaagcatta acatgaactt aaattcatca aggctaatct
     3061 ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt taataaccac tcataaatcc
     3121 tcatagagta tttgttttca aaagacttaa catgttccag attatatttt atgaattttt
     3181 ttaactggaa aagataaggc aatatctctt cactaaaaac taattctaat ttttcgcttg
     3241 agaacttggc atactttgtc cactggaaaa tctcaaagcc tttaaccaaa ggattcctga
     3301 tttccacagt tctcgtcatc agctctctgg ttgctttagc taatacacca taagcatttt
     3361 ccctactgat gttcatcatc tgagcgtatt ggttataagt gaacgatacc gtccgttctt
     3421 tccttgtagg gttttcaatc gtggggttga gtagtgccac acagcataaa attagcttgg
     3481 tttcatgctc cgttaagtca tagcgactaa tcgctagttc atttgctttg aaaacaacta
     3541 attcagacat acatctcaat tggtctaggt gattttaatc actataccaa ttgagatggg
     3601 ctagtcaatg ataattacta gtccttttcc tttgagttgt gggtatctgt aaattctgct
     3661 agacctttgc tggaaaactt gtaaattctg ctagaccctc tgtaaattcc gctagacctt
     3721 tgtgtgtttt ttttgtttat attcaagtgg ttataattta tagaataaag aaagaataaa
     3781 aaaagataaa aagaatagat cccagccctg tgtataactc actactttag tcagttccgc
     3841 agtattacaa aaggatgtcg caaacgctgt ttgctcctct acaaaacaga ccttaaaacc
     3901 ctaaaggctt aagtagcacc ctcgcaagct cgggcaaatc gctgaatatt ccttttgtct
     3961 ccgaccatca ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg
     4021 gctctggcag tgaatggggg taaatggcac tacataaggg attttgccga tttcggccta
     4081 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4141 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4201 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     4261 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     4321 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     4381 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     4441 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     4501 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     4561 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     4621 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     4681 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     4741 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     4801 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     4861 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     4921 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     4981 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     5041 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     5101 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5161 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5221 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5281 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     5341 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5401 tcctgttacc agtgcaggtc gactcgacaa ttcgcgcgcg aaggcgaagc ggcatgcatt
     5461 tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     5521 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     5581 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     5641 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     5701 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     5761 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     5821 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     5881 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     5941 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     6001 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     6061 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     6121 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     6181 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     6241 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     6301 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     6361 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     6421 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     6481 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     6541 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     6601 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt
     6661 gtcgagaaaa tttatcaaaa agagtgttga cttgtgagcg gataacaatg atacttagat
     6721 tcaattgtga gcggataaca atttcacaca
//
