LOCUS       sequence-404921-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy vector for expressing human DNA polymerase iota in E. coli 
            with an N-terminal 6x His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  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 7630)
  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: pJM919_WT His POLI
FEATURES             Location/Qualifiers
     source          1..7630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    401..417
                     /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    434..450
                     /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)."
     RBS             476..498
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             517..534
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             544..567
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2756..2773
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2826..2844)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2840..2887
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2924..3282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3011..3030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3221..3242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3297..3313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3297..3313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3321..3337
                     /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(3345..3351,3352..3369,3370..3375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3390..3411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3939..4889)
                     /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      4937..5159
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(5582..6397)
                     /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(5582..6397)
                     /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     6305..6324
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6389..6408
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        6806..6883
                     /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             join(6884..7630,1..336)
                     /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(6903..6922)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
       61 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
      121 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
      181 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
      241 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      301 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
      361 gcgcgaattg tcgagaaaat ttatcaaaaa gagtgttgac ttgtgagcgg ataacaatga
      421 tacttagatt caattgtgag cggataacaa tttcacacag aattctagaa ataattttgt
      481 ttaactttaa gaaggagata taacatgggc agcagccatc atcatcatca tcacagcagc
      541 ggcctggaag ttctgttcca ggggccccat atggaactgg cggatgttgg cgccgcggcg
      601 agcagccagg gcgttcatga tcaggtgtta ccaaccccga acgcgagcag ccgtgtgatt
      661 gtgcatgtgg atctggattg cttttatgcg caggtggaaa tgattagcaa cccggaactg
      721 aaagataaac cgctgggcgt gcagcagaaa tatctggtgg tgacctgcaa ctacgaggcc
      781 cgtaaactgg gcgtgaaaaa actgatgaac gtgcgtgatg cgaaagaaaa atgcccgcag
      841 ctggtgctgg tgaacggcga agatctgacc cgttatcgtg aaatgagcta taaagtgacc
      901 gaactgctgg aagaatttag cccggtggtg gaacgtctgg gctttgatga aaactttgtg
      961 gatctgaccg aaatggtgga aaaacgtctg cagcagctgc agagcgatga actgagcgcg
     1021 gtgaccgtga gcggccatgt gtataacaac cagagcatta acctgctgga tgtgctgcat
     1081 attcgtctgc tggtgggcag ccagattgcg gcggaaatgc gtgaagcgat gtataaccag
     1141 ctgggcctga ccggctgcgc gggcgtggcg agcaacaaac tgctggcgaa actggtgagc
     1201 ggcgtgttta aaccgaacca gcagaccgtg ctgctgccgg aaagctgcca gcatctgatt
     1261 catagcctga accatattaa agaaattccg ggcattggct ataaaaccgc gaaatgcctg
     1321 gaagcgctgg gcattaacag cgtgcgtgat ctgcagacct ttagcccgaa aattctggaa
     1381 aaagaactgg gcattagcgt ggcgcagcgt attcagaaac tgagctttgg cgaagataac
     1441 agcccggtga ttctgagcgg tccgccacaa agctttagcg aagaagatag ctttaaaaaa
     1501 tgcagcagcg aagtggaagc gaaaaacaaa attgaggaat tactggccag cctgctgaac
     1561 cgtgtgtgcc aggatggccg taaaccgcat accgtgcgtc tgattattcg tcgttatagc
     1621 agcgaaaaac attatggccg tgaaagccgt cagtgcccga ttccgagcca tgtgattcag
     1681 aaactgggca ccggcaacta tgatgtgatg accccgatgg tggatattct gatgaaactg
     1741 tttcgtaaca tggtgaacgt gaaaatgccg tttcatctga ccctgctgag cgtgtgcttt
     1801 tgcaacctga aagcgctgaa caccgcgaaa aaaggcctga ttgattatta tctgatgccg
     1861 agcctgagca ccaccagccg tagcggcaaa catagcttta aaatgaaaga tacccacatg
     1921 gaagattttc cgaaagataa agaaaccaac cgtgattttc tgccgagcgg ccgtattgaa
     1981 agcacccgta cccgtgaaag cccgctggat accaccaact ttagcaaaga aaaagatatt
     2041 aacgagtttc cgttatgctc tctgccggaa ggcgtggatc aggaagtgtt taaacagctg
     2101 ccggttgata tccaggaaga aattctgagc ggcaaaagcc gtgaaaaatt tcagggcaaa
     2161 ggcagcgtga gctgcccgct gcatgcgagc cgtggcgtgc tgagcttttt tagcaaaaaa
     2221 cagatgcagg atattccgat taacccgcgt gatcatctga gcagcagcaa acaggtgagc
     2281 agcgtgagcc cgtgcgaacc gggcaccagc ggctttaaca gcagcagcag cagctatatg
     2341 agcagccaga aagattatag ctattatctg gataaccgtc tgaaagatga acgtattagc
     2401 cagggcccga aagaaccgca gggctttcat tttaccaaca gcaacccggc ggtgagcgcc
     2461 ttccacagct ttccgaacct gcagagcgaa cagctgttta gccgtaacca tacgaccgat
     2521 agccataaac agaccgtggc gaccgatagc catgaaggcc tgaccgaaaa ccgtgaaccg
     2581 gatagcgtgg atgaaaaaat tacctttccg agcgatattg atccgcaggt gttttatgaa
     2641 ctgccggaag cggtgcagaa agaactgctg gcggaatgga aacgtaccgg cagcgatttt
     2701 catattggcc ataaataagt cggcctgcga attcgtcgac gcggccgcac tcgagcacca
     2761 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2821 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2881 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     2941 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3001 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3061 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3121 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3181 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3241 ggaacaacac tcaaccctat ctcggtctat tcttttgatt taaattcgta atcatggtca
     3301 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3361 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3421 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3481 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctatt tcttccagaa ttgccatgat
     3541 tttttcccca cgggaggcgt cactggctcc cgtgttgtcg gcagctttga ttcgataagc
     3601 agcatcgcct gtttcaggct gtctatgtgt gactgttgag ctgtaacaag ttgtctcagg
     3661 tgttcaattt catgttctag ttgctttgtt ttactggttt cacctgttct attaggtgtt
     3721 acatgctgtt catctgttac attgtcgatc tgttcatggt gaacagcttt aaatgcacca
     3781 aaaactcgta aaagctctga tgtatctatc ttttttacac cgttttcatc tgtgcatata
     3841 tggacagttt tccctttgat atgtaacggt gaacagttgt tctacttttg tttgttagtc
     3901 ttgatgcttc actgatagat acaagagcca taagaacctc agatccttcc gtatttagcc
     3961 agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga
     4021 tcatacttac tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt
     4081 gcagttaaag catcgtgtag tgtttttctt agtccgttat gtaggtagga atctgatgta
     4141 atggttgttg gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg
     4201 agatccattt gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc
     4261 ttatcaacca ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa
     4321 acccattggt taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta
     4381 aattcatcaa ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt
     4441 aataaccact cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga
     4501 ttatatttta tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact
     4561 aattctaatt tttcgcttga gaacttggca tactttgtcc actggaaaat ctcaaagcct
     4621 ttaaccaaag gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct
     4681 aatacaccat aagcattttc cctactgatg ttcatcatct gagcgtattg gttataagtg
     4741 aacgataccg tccgttcttt ccttgtaggg ttttcaatcg tggggttgag tagtgccaca
     4801 cagcataaaa ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca
     4861 tttgctttga aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca
     4921 ctataccaat tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg
     4981 ggtatctgta aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct
     5041 gtaaattccg ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat
     5101 agaataaaga aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca
     5161 ctactttagt cagttccgca gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta
     5221 caaaacagac cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg
     5281 ctgaatattc cttttgtctc cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat
     5341 tcagttcgct gcgctcacgg ctctggcagt gaatgggggt aaatggcact acataaggga
     5401 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5461 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     5521 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     5581 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     5641 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     5701 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     5761 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     5821 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     5881 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     5941 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     6001 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     6061 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     6121 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     6181 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     6241 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     6301 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     6361 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     6421 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6481 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6541 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6601 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6661 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6721 acatacctcg ctctgctaat cctgttacca gtgcaggtcg actcgacaat tcgcgcgcga
     6781 aggcgaagcg gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat
     6841 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     6901 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     6961 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     7021 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     7081 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     7141 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     7201 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     7261 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     7321 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     7381 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     7441 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     7501 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     7561 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     7621 cgatcagatg
//
