LOCUS       sequence-352734-        5137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology region +/- 800bp to the transcription start site of POLB, 
            with EGFP inserted in-frame on the N-terminus of POLB, and a 
            mutation in the PAM site used by POLBKO gRNA1 in POLB exon1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5137)
  AUTHORS   Koczor CA, Saville KM, Andrews JF, Clark J, Fang Q, Li J, 
            Al-Rahahleh RQ, Ibrahim M, McClellan S, Makarov MV, Migaud ME, Sobol
            RW
  TITLE     Temporal dynamics of base excision/single-strand break repair 
            protein complex assembly/disassembly are modulated by the 
            PARP/NAD(+)/SIRT6 axis.
  JOURNAL   Cell Rep. 2021 Nov 2;37(5):109917. doi: 
            10.1016/j.celrep.2021.109917.
  PUBMED    34731617
REFERENCE   2  (bases 1 to 5137)
  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: "Cell Rep.";
            date: "2021-11-2"; volume: "37(5):109917. doi"; pages: " 
            10.1016/j.celrep.2021.109917"
COMMENT     Sequence Label: pUC19-POLBHR-eGFP
FEATURES             Location/Qualifiers
     source          1..5137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     regulatory      1143..1152
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1149..1151,1152..1154,1155..1865)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1149..1151,1152..1154,1155..1865)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1194..1215)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1455..1474)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1802..1823
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2741..2758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2741..2757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2750..2772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2966..2985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3085..3107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3145..3163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3231..3335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3336..3404,3405..4196)
                     /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     complement(3554..3573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4367..4955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4856..4875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5109..5126
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttat
      241 tgcagttcga tatgaaccca ggagttacgc ttgtaaaata aactattgct gatgtgacat
      301 ttccatttct caagcagact gtaaatttct cagatgtagg tcatcatgtt ataattaggg
      361 ttccattgct catgatctag atgaaacact tctccctttc ttgtggtctc ttcattaatt
      421 tggtccctac cgcaaaggca gggggattaa aaataaaata tcttttgaca ctggttggca
      481 ggtgtactaa cacgtgtgcg ggaggattcc tgttctcggc atggttcacg ccgggctccg
      541 gcagcgccct agtacgtaag gggctgcact ggagtgtgga ctgcccggta tccttagaga
      601 aagttttgac agtgtgacgc cagtgtcgcc tctaaccacg tacaaaacgc gccatgcatt
      661 gggtagctat tacagtgcac atttttgtgc aggaagctac cccaccagcg caagtatttc
      721 agaatcaaga tcgcactccc gtttcccctt ctagggaaag gattccagat aaacactgag
      781 aaaacagatt taatatctta ggtcaaaact ccaggtcttc ccataggaag gcccctgggc
      841 tgtcattctg aggtgcctat ttcccccgcg ccgtgtccac gtccacgagt ccacgaaccc
      901 tccgagttct ctctcctcac ctgccgaggg ctcgcccctc tagccccgcc ccgccccgcc
      961 caggacgcgt gacgtcacaa caagcgccgt gcgccccgcc ccccatcggg ggcaaccatt
     1021 gttccgccgg tcgcgccgga gctgggttgc tcctgctccc gtctccaagt cctggtacct
     1081 ccttcaagct gggagagggc tctagtccct ggttctgaac actctggggt tctcgggtgc
     1141 aggccgccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1201 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1261 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1321 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1381 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1441 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1501 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1561 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1621 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1681 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1741 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1801 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1861 acaagatgag caaacggaag gcgccgcaag agactctcaa cgggggaatc accgacatgc
     1921 tcacaggtta gcaccgggcc gggccccgct ggctttcttc tttccttcca gcctcttccc
     1981 ctgccttcct tctctcccac accgacagtc cagtgggtag ggtaggttcc ttgcagcggg
     2041 tcgtcttccg tggggatctc cctccggcgc ccctggctgg ttgtcagtcc tgccggctac
     2101 acctgggcca tcgcttgggc tgcttttggt ctggcccttg gaggaaacgg gtggtcactg
     2161 tcttttagag gctgccatat ccccttccag aaaacagttc tcgtttgttt actcgtggtt
     2221 cttgttcacc cgagccttct gttgcctttc agaactcgca aactttgaga agaacgtgag
     2281 ccaagctatc cacaagtaca atgcttacag gtgggacagt gcagcattct cgggtagcat
     2341 acgttctggg ataccctgtt tagtgtggca attaacagga ctgagggccc agtggatatt
     2401 tggtccatct gcaagagcgg gaaaaagcaa gaatcgaggc tggtacctta ctatttcttg
     2461 aagaatgtgg gcagtgcgtt ataggatcag ttcagataag ttcccaattc tgattgcaga
     2521 tagctgtagc ctgatacgta ttttgtcatc atcaaacatg aattactaga aatgcggtgt
     2581 aattttaacc gtaatgaaag acagcggatg aagtccatac tgtgaaatat atgtggttca
     2641 aggaatcaca ttttttcttt tggttggtct aaagttgatc ctgtgggatc ctgatcctga
     2701 tccttgttcc ttgttcatgg gatcctgtcc ttgtgaattc actggccgtc gttttacaac
     2761 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2821 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2881 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2941 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3001 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3061 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3121 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3181 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3241 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3301 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3361 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3421 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3481 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3541 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3601 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3661 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3721 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3781 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3841 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3901 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3961 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4021 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4081 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4141 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4201 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4261 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4321 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4381 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4441 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4501 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4561 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4621 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4681 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4741 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4801 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4861 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4921 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4981 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5041 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5101 ccgagcgcag cgagtcagtg agcgaggaag cggaaca
//
