LOCUS       sequence-435332-        5767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of cysteine-free variant of the E. coli 
            ribonuclease HI gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5767)
  AUTHORS   Bhattacharya S, Varney KM, Dahmane T, Johnson BA, Weber DJ, Palmer 
            AG 3rd
  TITLE     Deuterium spin relaxation of fractionally deuterated ribonuclease H 
            using paired 475 and 950 MHz NMR spectrometers.
  JOURNAL   J Biomol NMR. 2024 Jun 10. doi: 10.1007/s10858-024-00443-w.
  PUBMED    38856928
REFERENCE   2  (bases 1 to 5767)
  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: "J Biomol 
            NMR. 2024 Jun 10. doi: 10.1007/s10858-024-00443-w."
COMMENT     Sequence Label: pMCSG7-10xHis-ecRNHI*
FEATURES             Location/Qualifiers
     source          1..5767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             631..822
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(816..838)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    924..1063
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     979..998
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1078..1095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1249..1837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1329..1348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2008..2799,2800..2868))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2631..2650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2869..2972)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3139..3158
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3282..3329)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3325..3343
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3396..3413)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3951..3971)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(3996..4025)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4036..4058
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4073..4097
                     /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     complement(4097..4116)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4098..4116)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4204..4223)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4429..4506
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4507..5589
                     /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     complement(4526..4545)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
       61 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      121 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      181 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      241 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      301 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
      361 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
      421 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
      481 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
      541 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
      601 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
      661 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
      721 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
      781 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
      841 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
      901 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
      961 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1021 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1081 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1141 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1201 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1261 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1321 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1381 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1441 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1501 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1561 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1621 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1681 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1741 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1801 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1861 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1921 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1981 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2041 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2101 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2161 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2221 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2281 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     2341 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2401 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2461 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2521 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2581 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2641 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2701 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2761 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2821 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2881 tcaaattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2941 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     3001 ttgtaagcgt taatgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3061 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3121 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3181 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3241 cgcgtcccat tcgccaatcc ggatatagtt cctcctttca gcaaaaaacc cctcaagacc
     3301 cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc agccaactca
     3361 gcttcctttc gggctttgtt agcagccgga tctcagtggt ggtggtggtg gtgctcgagt
     3421 gcggccgcaa gcttgtcgac ggagctcgaa ttcggatccg ttatccactt ccaatgtcaa
     3481 acttcaactt gataccctgt atcttccagt gtgggattca tcgctgcggc gcgcgctagc
     3541 tcatctgcgc gttcgttttc cgggtgaccg gcatggcctt taacccattc ccatttgatt
     3601 tgatgctgcc ctagggcagc atcaagacgt tgccagagat ctacattttt tactggtttt
     3661 ttgtccgcgg ttttccagcc acgttttttc cagttatgga tccactgggt gataccttgg
     3721 cggacatact ggctgtcggt cgacaaaatg acttcggcat gttcttttaa cgcctccagc
     3781 gcgacaatag cggccatgag ctccatacgg ttgttggtgg tacgcgtgta gcctgcagaa
     3841 aaggttttct cgcggccgcg atagcgtaaa atggcgccgt aacccccggg ccctggattg
     3901 cccagagccg aaccatcggt gaaaatttct acctgtttaa gcatggcatt ggattggaag
     3961 tacaggttct cggtacccag atctacacca gaagagtgat ggtggtgatg gtgatggtga
     4021 tgatgcatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
     4081 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct
     4141 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
     4201 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     4261 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     4321 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     4381 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
     4441 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     4501 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     4561 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     4621 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     4681 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     4741 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     4801 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     4861 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     4921 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     4981 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     5041 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     5101 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     5161 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     5221 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     5281 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     5341 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     5401 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     5461 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     5521 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     5581 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     5641 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     5701 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     5761 agcgctc
//
