LOCUS       sequence-256737-        6253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of bacterial RNase HI tagged with NLS-mCherry
            that can be used to degrade R-loops. Confers resistance to 
            Puromycin. Expression is inducible in T-REx cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  AUTHORS   Britton S, Dernoncourt E, Delteil C, Froment C, Schiltz O, Salles B,
            Frit P, Calsou P
  TITLE     DNA damage triggers SAF-A and RNA biogenesis factors exclusion from 
            chromatin coupled to R-loops removal.
  JOURNAL   Nucleic Acids Res. 2014 Oct 1;42(14):9047-62. doi: 
            10.1093/nar/gku601. Epub 2014 Jul 16.
  PUBMED    25030905
REFERENCE   2  (bases 1 to 6253)
  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"; doi: 
            "10.1093/nar/gku601"; journalName: "Nucleic Acids Res"; date: 
            "2014-10-1- 1"; volume: "42"; issue: "14"; pages: "9047-62"
COMMENT     Sequence Label: pICE-RNaseHI-WT-NLS-mCherry
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1210..1230
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1243..1263
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1273..1282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1279..1989
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1424..1442)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1692..1711
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2057..2074)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2063..2287
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2333..2761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2420..2439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2630..2651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2770..2790)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2775..3104
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2955..3090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3017..3036
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3156..3755
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3156..3175)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3652..3672
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3885..4006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3922..3941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3976..3995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4056..4072)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4056..4072)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4069..4091)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4080..4096
                     /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(4104..4110,4111..4128,4129..4134))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4149..4170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4287..4304)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4458..5046)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4538..4557)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5217..6008,6009..6077))
                     /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     5840..5859
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6078..6182)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggcaagc ttgccaccat gctgaaacag gtggaaatct tcaccgacgg cagctgtctg
      781 ggcaatcctg gccctggcgg atacggcgcc atcctgagat accggggcag agagaaaacc
      841 ttcagcgccg gctacacccg gaccaccaac aaccggatgg agctgatggc cgccatcgtg
      901 gccctggaag ccctgaaaga acactgcgaa gtgatcctga gcaccgacag ccagtacgtg
      961 cggcagggca tcacccagtg gattcacaac tggaagaagc ggggctggaa aaccgccgac
     1021 aagaaacccg tgaagaacgt ggacctgtgg cagagactgg atgccgccct gggacagcac
     1081 cagatcaagt gggagtgggt caagggccac gccggccacc ctgagaatga gagatgtgac
     1141 gagctggcca gagccgccgc tatgaacccc accctggaag ataccggcta ccaggtggaa
     1201 gtgggcagcc ccaagaaaaa gcggaaagtg ggcacgcgtg gccccaaaaa gaaacgcaaa
     1261 gtgggaccgg tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     1321 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     1381 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     1441 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     1501 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     1561 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     1621 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     1681 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     1741 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     1801 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     1861 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     1921 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     1981 tacaagtaaa gcggccgcga attcggatcc ggtaccctcg agacgcgtct gcagtctaga
     2041 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2101 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2161 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2221 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2281 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2341 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2401 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2461 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2521 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2581 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2641 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2701 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2761 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2821 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2881 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2941 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3001 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3061 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3121 gtatatccat tttcggatct gatcagcacg tgaccatgac cgagtacaag cccacggtgc
     3181 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     3241 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     3301 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     3361 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     3421 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     3481 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     3541 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     3601 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     3661 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     3721 gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat ttcgattcca
     3781 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3841 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     3901 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3961 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctag
     4021 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4081 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4141 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4201 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4261 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4321 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4381 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4441 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4501 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4561 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4621 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4681 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4741 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4801 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4861 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4921 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4981 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5041 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5101 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5161 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5221 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5281 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5341 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5401 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5461 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5521 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5581 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5641 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5701 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5761 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5821 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5881 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5941 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6001 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6061 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6121 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6181 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcc tgtacgggcc
     6241 agatatccgc gtt
//
