LOCUS       sequence-456413-        6253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of an inactive mutant of bacterial RNase HI 
            tagged with NLS-mCherry that can be used to detect 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-D10R-E48R-NLS-mCherry
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             483..503
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             516..536
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      546..555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             552..1262
                     /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(697..715)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     965..984
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1330..1347)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1336..1560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1606..2034
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1693..1712)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1903..1924
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2043..2063)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2048..2377
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2228..2363
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2290..2309
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2429..3028
                     /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(2429..2448)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2925..2945
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3158..3279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3195..3214)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3249..3268
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3329..3345)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3329..3345)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3342..3364)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3353..3369
                     /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(3377..3383,3384..3401,3402..3407))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3422..3443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3560..3577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3731..4319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3811..3830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4490..5281,5282..5350))
                     /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     5113..5132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5351..5455)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5527..5906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5907..6110
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6061..6081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6112..6130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6133..6151
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6161..6185
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 agcttgccac catgctgaaa caggtggaaa tcttcacccg gggcagctgt ctgggcaatc
       61 ctggccctgg cggatacggc gccatcctga gataccgggg cagagagaaa accttcagcg
      121 ccggctacac ccggaccacc aacaaccgga tgagactgat ggccgccatc gtggccctgg
      181 aagccctgaa agaacactgc gaagtgatcc tgagcaccga cagccagtac gtgcggcagg
      241 gcatcaccca gtggattcac aactggaaga agcggggctg gaaaaccgcc gacaagaaac
      301 ccgtgaagaa cgtggacctg tggcagagac tggatgccgc cctgggacag caccagatca
      361 agtgggagtg ggtcaagggc cacgccggcc accctgagaa tgagagatgt gacgagctgg
      421 ccagagccgc cgctatgaac cccaccctgg aagataccgg ctaccaggtg gaagtgggca
      481 gccccaagaa aaagcggaaa gtgggcacgc gtggccccaa aaagaaacgc aaagtgggac
      541 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
      601 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
      661 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      721 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
      781 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
      841 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
      901 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
      961 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1021 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1081 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     1141 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1201 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     1261 aaagcggccg cgaattcgga tccggtaccc tcgagacgcg tctgcagtct agataaaccc
     1321 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1381 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1441 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1501 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     1561 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     1621 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1681 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1741 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1801 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1861 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1921 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1981 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2041 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2101 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2161 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2221 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2281 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     2341 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     2401 cattttcgga tctgatcagc acgtgaccat gaccgagtac aagcccacgg tgcgcctcgc
     2461 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2521 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     2581 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     2641 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     2701 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     2761 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     2821 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     2881 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     2941 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3001 gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga gatttcgatt ccaccgccgc
     3061 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3121 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3181 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3241 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tagccgtcga
     3301 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3361 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3421 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3481 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3541 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3601 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3661 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3721 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3781 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3841 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3901 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3961 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4021 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4081 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4141 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4201 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4261 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4321 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4381 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4441 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4501 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4561 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4621 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4681 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4741 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4801 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4861 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4921 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4981 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5041 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5101 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5161 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5221 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5281 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5341 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5401 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5461 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcctgtacgg gccagatatc
     5521 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     5581 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     5641 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     5701 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     5761 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     5821 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     5881 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     5941 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     6001 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     6061 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
     6121 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
     6181 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     6241 ccagcctccg gca
//
