LOCUS       sequence-341899-        6855 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express GFP-tagged RNase H1 WT in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6855)
  AUTHORS   Crossley MP, Brickner JR, Song C, Zar SMT, Maw SS, Chedin F, Tsai 
            MS, Cimprich KA
  TITLE     Catalytically inactive, purified RNase H1: A specific and sensitive 
            probe for RNA-DNA hybrid imaging.
  JOURNAL   J Cell Biol. 2021 Sep 6;220(9). pii: 212458. doi: 
            10.1083/jcb.202101092. Epub 2021 Jul 7.
  PUBMED    34232287
REFERENCE   2  (bases 1 to 6855)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2021-09-6"; volume: "220(9). pii: 212458. doi"; 
            pages: " 10.1083/jcb.202101092. Epub 2021 Jul 7"
COMMENT     Sequence Label: 1GFP/RNase H1 WT
FEATURES             Location/Qualifiers
     source          1..6855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(696..1778)
                     /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     1740..1759
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1779..1856)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2062..2081
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2165..2184
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2165..2183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2184..2208
                     /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             2223..2245
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2264..2281
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(2291..2293,2294..3004)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2333..2354)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2941..2962
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3014..3034
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3883..3900
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3953..3971)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3967..4014
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4051..4506
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4138..4157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4348..4369
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4599..5414)
                     /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(4599..5414)
                     /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     5322..5341
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5406..5425
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5536..6124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6025..6044
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6278..6295
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6310..6449
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6375..6394)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6535..6557
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6551..6742)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
       61 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
      121 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
      181 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
      241 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
      301 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
      361 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
      421 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
      481 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
      541 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
      601 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
      661 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      721 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      781 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      841 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      901 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      961 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1021 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc
     1081 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1141 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1201 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1261 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1321 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1381 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1441 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1501 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1561 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1621 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1681 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1741 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1801 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1861 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1921 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1981 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     2041 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     2101 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     2161 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     2221 attttgttta actttaagaa ggagatatac catgggttct tctcaccatc accatcacca
     2281 tggttcttct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2341 gctggacggc gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc
     2401 caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2461 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2521 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2581 catctccttc aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga
     2641 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2701 ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca
     2761 gaagaacggc atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca
     2821 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2881 caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca
     2941 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3001 caaggggatc gaggaaaacc tgtacttcca atccaatatg ttctatgccg tgaggagggg
     3061 ccgcaagacc ggggtctttc tgacctggaa tgagtgcaga gcacaggtgg accggtttcc
     3121 tgctgccaga tttaagaagt ttgccacaga ggatgaggcc tgggcctttg tcaggaaatc
     3181 tgcaagcccg gaagtttcag aagggcatga aaatcaacat ggacaagaat cggaggcgaa
     3241 agccagcaag cgactccgtg agccactgga tggagatgga catgaaagcg cagagccgta
     3301 tgcaaagcac atgaagccga gcgtggagcc ggcgcctcca gttagcagag acacgttttc
     3361 ctacatggga gacttcgtcg tcgtctacac tgatggctgc tgctccagta atgggcgtag
     3421 aaggccgcga gcaggaatcg gcgtttactg ggggccgggc catcctttaa atgtaggcat
     3481 tagacttcct gggcggcaga caaaccaaag agcggaaatt catgcagcct gcaaagccat
     3541 tgaacaagca aagactcaaa acatcaataa actggttctg tatacagaca gtatgtttac
     3601 gataaatggt ataactaact gggttcaagg ttggaagaaa aatgggtgga agacaagtgc
     3661 agggaaagag gtgatcaaca aagaggactt tgtggcactg gagaggctta cccaggggat
     3721 ggacattcag tggatgcatg ttcctggtca ttcgggattt ataggcaatg aagaagctga
     3781 cagattagcc agagaaggag ctaaacaatc ggaagactaa attggaagtg gataacggat
     3841 ccgaattcga gcgccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac
     3901 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     3961 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     4021 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     4081 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4141 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4201 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4261 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4321 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4381 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4441 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaacta gtaacgttta
     4501 caatttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4561 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     4621 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     4681 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     4741 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     4801 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     4861 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     4921 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     4981 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     5041 gccagcgcat caacaatgtt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     5101 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     5161 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     5221 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     5281 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     5341 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     5401 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     5461 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5521 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5581 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5641 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5701 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5761 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5821 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5881 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5941 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6001 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6061 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6121 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6241 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6301 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     6361 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     6421 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     6481 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6541 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     6601 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     6661 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     6721 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     6781 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     6841 cccggttact ggaac
//
