LOCUS       sequence-381766-        7924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered PcrA_M6 helicase to support isothermal amplification 
            method SHARP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7924)
  AUTHORS   Gavrilov M, Yang JYC, Zou RS, Ma W, Lee CY, Mohapatra S, Kang J, 
            Liao TW, Myong S, Ha T
  TITLE     Engineered helicase replaces thermocycler in DNA amplification while
            retaining desired PCR characteristics.
  JOURNAL   Nat Commun. 2022 Oct 23;13(1):6312. doi: 10.1038/s41467-022-34076-0.
  PUBMED    36274095
REFERENCE   2  (bases 1 to 7924)
  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: "Nat 
            Commun."; date: "2022-10-23"; volume: "13(1):6312. doi"; pages: " 
            10.1038/s41467-022-34076-0"
COMMENT     Sequence Label: pET-11b_PcrA_M6_H93A
FEATURES             Location/Qualifiers
     source          1..7924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2300..2371
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(2425..2443)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2439..2486
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2661..2666,2667..2683,2684..2689))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2716..2734)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2802..2906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2907..2975,2976..3767)
                     /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(3125..3144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3938..4526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4427..4446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4680..4697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4712..4851
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4777..4796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4937..4959
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4953..5144)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6456..7538)
                     /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     7500..7519
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7539..7616)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7822..7841
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcatcaccat caccatcacg ggtccggaat
      121 gaacttttta tcggaacagc tgctcgccca tttaaacaaa gagcaacaag aagccgtcag
      181 gacgacggaa ggcccgctgc tcattatggc gggggcggga agcgggaaaa cgcgggtgtt
      241 gacgcaccgc atcgcctatt tgatggcgga aaagcatgtg gcgccgtgga acattttggc
      301 cattacgttt acgaacaagg cggcgcgcga aatgcgggaa cgtgtgcagt cgctcttagg
      361 tggggcggcg gaagacgtct ggatttcgac gttcgcgtcg atggccgtcc gcattttgcg
      421 ccgcgacatt gaccgcatcg gcatcaaccg caatttttcc atccttgatc cgacggacca
      481 gctttcagtc atgaaaacga ttttaaaaga aaaaaacata gacccgaaaa aatttgagcc
      541 gcggacgatt ttaggcacga tcagcgcggc gaaaaacgag ctgttgcctc cggagcaatt
      601 cgcgaagcgg gcctcgacgt attacgaaaa agtcgtcagc gatgtgtatc aagaatacca
      661 acagcgcctg cttcgctgtc attcgctcga ttttgacgat ttgatcatga cgacgatcca
      721 actgtttgac cgcgtgccgg atgtgcttca ctattaccaa tataagtttc agtacattca
      781 tattgatgag taccaggata cgaaccgcgc tcaatatacg ctggtcaaaa agctggcgga
      841 acgctttcaa aacattgccg ccgtcggcga cgccgaccaa tcgatttatc ggtggcgcgg
      901 ggcggacatc caaaacattt tgtcgttcga gcgcgactat ccgaacgcaa aagtcatttt
      961 gcttgaacaa aactaccgct cgacgaagcg cattttgcaa gcggcgaacg aagtcatcga
     1021 gcataacgtc aaccggaagc cgaaacggct ttggacggaa aacccggaag gaaagccgat
     1081 tctttattat gaggcgatga acgaagcgga cgaagcgcag tttgtcgctg gacgcatccg
     1141 cgaggcggtg gagcgcggcg aacgccgcta ccgtgatttt gctgtcttgt accggacgaa
     1201 cgcccagtcg cgtgtcatgg aggaaatgtt gctgaaagcg aacattccgt atcaaattgt
     1261 cggcggcgta aagttctatg accggaaaga aattaaagac attctcgcct atttgcgcgt
     1321 cattgccaat ccggacgatg attgcagctt gcttcgcatc attaacgtgc caaaacgcgg
     1381 cattggcgcc tcgacgatcg acaaactcgt ccgctatgca gccgatcatg agctgtcctt
     1441 gtttgaggcg ctcggcgagc tagagatgat cgggcttggc gccaaagcgg ccggggcgct
     1501 cgccgcgttc cgcagccagc tcgagcaatg gacacagctg caagaatacg tctccgtcac
     1561 cgaactcgtc gaagaagtgc tcgacaaatc gggctaccgc gagatgctca aggcggagcg
     1621 gacgattgaa gcacaaagcc ggctcgagaa cttggatgag tttttgtcgg tgacgaagca
     1681 ttttgaaaat gtgagcgacg ataaatcgct catcgccttt ttaaccgact tggcgctcat
     1741 ttccgatttg gacgagctga acgggacgga acaggccgct gaaggagatg ccgtcatgtt
     1801 gatgacgttg catgccgcca aagggctcga gtttccggtc gtctttttga tcggcatgga
     1861 agaaggcatt ttcccgcaca accgctctct cgaggatgac gatgagatgg aagaagaacg
     1921 gcggctggcg tacgtcggca tcacccgcgc ggaggaagaa cttgtgctga cgagcgcgca
     1981 aatgcggacg ttgtttggca acatccaaat gaacccgccg tcgcgctttt tgaatgaaat
     2041 tccggcgcat ttgcttgaga cagcctcgcg ccgccaagcg ggcgcctccc gcccggccgt
     2101 ttcgcgcccg caggcaagcg gcgccgtggg atcgtggaaa gtcggcgatc gggcgaatca
     2161 ccggaaatgg ggcatcggca ccgtcgtcag cgtccgcggc ggcggcgacg accaagagct
     2221 cgacatcgcc ttcccgagcc cgatcggcat taaacggttg cttgccaaat ttgcgccgat
     2281 tgagaaagtg ggtagcggcg actacaagga cgatgacgat aaggattata aagacgatga
     2341 cgataaggac tacaaagacg atgacgataa atagggatcc ggctgctaac aaagcccgaa
     2401 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2461 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     2521 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     2581 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     2641 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt
     2701 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2761 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2821 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2881 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2941 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3001 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3061 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3121 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     3181 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3241 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3301 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3361 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3421 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     3481 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3541 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3601 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3661 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3721 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3781 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3841 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4021 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4081 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4561 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4621 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4681 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4741 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4801 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4861 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4921 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4981 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5041 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5101 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5161 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5221 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5281 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5341 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5401 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5461 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5521 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5581 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     5641 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     5701 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     5761 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     5821 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     5881 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     5941 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     6001 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     6061 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     6121 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     6181 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     6241 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     6301 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     6361 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     6421 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6481 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6541 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     6601 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     6661 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6721 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6781 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6841 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     6901 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     6961 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     7021 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     7081 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     7141 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     7201 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     7261 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     7321 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     7381 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     7441 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     7501 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     7561 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     7621 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     7681 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     7741 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     7801 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     7861 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     7921 aaat
//
