LOCUS       sequence-338468-        5364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tandem repeats of HBD domain of RNase H1 for better 
            DNA-RNA Hybrid recognition specificity in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5364)
  AUTHORS   Wang K, Wang H, Li C, Yin Z, Xiao R, Li Q, Xiang Y, Wang W, Huang J,
            Chen L, Fang P, Liang K
  TITLE     Genomic profiling of native R loops with a DNA-RNA hybrid 
            recognition sensor.
  JOURNAL   Sci Adv. 2021 Feb 17;7(8). pii: 7/8/eabe3516. doi: 
            10.1126/sciadv.abe3516. Print 2021 Feb.
  PUBMED    33597247
REFERENCE   2  (bases 1 to 5364)
  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: "Sci Adv."; 
            date: "2021-02-17"; volume: "7(8). pii: 7/8/eabe3516. doi"; pages: "
            10.1126/sciadv.abe3516. Print 2021 Feb"
COMMENT     Sequence Label: pGEX-GST-His6-2_HBD
FEATURES             Location/Qualifiers
     source          1..5364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(531..536,537..554,555..561)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    569..585
                     /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     593..609
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     593..609
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             605..1132
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(625..642)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(625..641)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(634..656)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1234..1239,1240..1255,1256..1262)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1270..1286
                     /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)."
     CDS             1309..1962
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1920..1942
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1969..1986
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2017..2046
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2465..2487)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2632..2650)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2718..2822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2823..2891,2892..3683)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3041..3060)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3854..4442
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4343..4362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4596..4613
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4686..4763
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4764..5364,1..482)
                     /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(4783..4802)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccccaggc tttacacttt
      541 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      601 agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg actgggaaaa
      661 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
      721 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
      781 gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct
      841 tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc
      901 catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa
      961 tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca
     1021 gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt gcaccaatgc
     1081 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     1141 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     1201 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
     1261 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat gtcccctata
     1321 ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt
     1381 gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa
     1441 aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa
     1501 ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
     1561 tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac
     1621 ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc
     1681 aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat
     1741 ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac
     1801 atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
     1861 gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag
     1921 ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct ggttccgcgt
     1981 ggatctcgtc gtgcatctgt tggatatacc atgggccatc atcatcatca tcatcatcat
     2041 catcacagca gcggccatat cgaagatcgt catatgatgt tctatgccgt gaggaggggc
     2101 cgcaagaccg gggtctttct gacctggaat gagtgcagag cacaggtgga ccggtttcct
     2161 gctgccagat ttaagaagtt tgccacagag gatgaggcct gggcctttgt caggaaatct
     2221 gcaagcggag gcggaggggg aggaatgttc tatgccgtga ggaggggccg caagaccggg
     2281 gtctttctga cctggaatga gtgcagagca caggtggacc ggtttcctgc tgccagattt
     2341 aagaagtttg ccacagagga tgaggcctgg gcctttgtca ggaaatctgc aagcggaggc
     2401 ggagggggag gaattcatcg tgactgacga tctgcctcgc gcgtttcggt gatgacggtg
     2461 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2521 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     2581 tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga
     2641 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2701 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2761 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2821 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2881 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2941 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3001 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3061 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3121 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3181 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3241 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3301 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3361 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3421 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3481 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3541 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3601 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3661 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3721 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3781 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3841 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3901 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3961 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4021 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4081 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4141 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4201 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4261 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4321 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4381 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4441 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4501 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4561 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4621 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     4681 attccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     4741 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     4801 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     4861 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     4921 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     4981 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     5041 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     5101 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     5161 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     5221 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     5281 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     5341 cggcgcgtct gcgtctggct ggct
//
