LOCUS       sequence-481872-        6375 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Expression of the engineered N-terminal hybrid-binding domain of 
            RNase H3 (enDR3) for capturing R-loops. A TEV cleavage site, mEGFP 
            and nuclear localization signal (NLS) are included in the fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6375)
  TITLE     Genome-wide in vivo and ex vivo mapping of R-loops using engineered 
            N-terminal hybrid-binding domain of RNase H3 (enDR3)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6375)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: enDR3-TEV-mEGFP-NLS (pRS1451)
FEATURES             Location/Qualifiers
     source          1..6375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1002..1022
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1458..1478
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1485..1487,1488..1490,1491..2201)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1530..1551)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1791..1810)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2138..2159
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2214..2234
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2238..2258
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2262..2282
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2327..2344)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2333..2557
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2690..2709)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2841..2888
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2896..3918
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2896..3918
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4048..4169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4085..4104)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4139..4158
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4218..4234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4218..4234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4231..4253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4242..4258
                     /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(4266..4272,4273..4290,4291..4296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4311..4332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4449..4466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4620..5208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4700..4719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5379..6170,6171..6239))
                     /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     6002..6021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6240..6344)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctggcgtt taaacttaag cttggtaccg agctcggatc cgaaaacctg tacttccaag
     1021 gaaccggtat gagcaactac gtgatccagg ccgaccaaca gctgctggat gcgcttcgtg
     1081 cgcactacga aggtgcgctg tctgaccgcc tgccggctgg cgctctgttc gcggttaaac
     1141 gtccggatgt tgtgattacc gcatatcgtt ccggtaaagt aatgtttcag ggtaaggccg
     1201 ctgagcagga agcagctaag tggatctcgg gtggctcagg cggctctggc ggttctaact
     1261 acgttatcca ggcagaccag cagctgctgg acgcgctgcg tgcgcactac gaaggagcgc
     1321 tgtcggaccg tctgccggcg ggcgcactgt tcgcggttaa acgtccggac gttgtaatca
     1381 ccgcgtaccg ttccggtaaa gttatgttcc agggtaaagc ggcggaacag gaagcggcga
     1441 aatggatctc tgctagcgag aatttgtatt ttcagggtga tatcatggtg agcaagggcg
     1501 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1561 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1621 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1681 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1741 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1801 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1861 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1921 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1981 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2041 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2101 ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2161 ccgccgccgg gatcactctc ggcatggacg agctgtacaa ggccgcagca gaccccaaga
     2221 aaaagcggaa ggtggaccct aaaaagaaga gaaaggtcga cccaaagaag aaacgcaagg
     2281 tgtaagtaac tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     2341 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2401 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2461 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2521 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     2581 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2641 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2701 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2761 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2821 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
     2881 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
     2941 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
     3001 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
     3061 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
     3121 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
     3181 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
     3241 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     3301 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     3361 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     3421 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     3481 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     3541 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     3601 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     3661 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     3721 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     3781 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     3841 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     3901 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     3961 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4021 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4081 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4141 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4201 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4261 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4321 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4381 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4441 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4501 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4561 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4621 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4681 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4741 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4801 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4861 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4921 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4981 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5041 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5101 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5161 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5221 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5281 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5341 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5401 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5461 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5521 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5581 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5641 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5701 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5761 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5821 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5881 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5941 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6001 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6061 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6121 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6181 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6241 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6301 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6361 gtgccacctg acgtc
//
