LOCUS       sequence-406202-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bst polymerase large fragment (BF) F710Y/D598A, His6-tagged, HRV 
            3C-cleavable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7101)
  AUTHORS   Lelyveld VS, Fang Z, Szostak JW
  TITLE     Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase 
            activity.
  JOURNAL   Science. 2023 Oct 27;382(6669):423-429. doi: 
            10.1126/science.adh5339. Epub 2023 Oct 26.
  PUBMED    37883544
REFERENCE   2  (bases 1 to 7101)
  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"; doi: 
            "10.1126/science.adh5339"; journalName: "Science"; date: 
            "2023-10-27- 27"; volume: "382"; issue: "6669"; pages: "423-429"
COMMENT     Sequence Label: pVSL10
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(115..129,130..135,136..152,153..159)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    135..151
                     /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)."
     protein_bind    167..183
                     /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             202..213
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             232..249
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             259..282
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2248..2267)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2473..2550
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             2551..3633
                     /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(2570..2589)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4442..4633
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4627..4649)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4735..4874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4790..4809
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4889..4906)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5060..5648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5140..5159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5759..5778)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5770..6585
                     /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             5770..6585
                     /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     complement(5843..5862)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6677..7101,1..31))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6814..6835)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7026..7045
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc
       61 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aagggctcga gaaatcataa
      121 aaaatttatt tgctttgtga gcggataaca attataatag attcaattgt gagcggataa
      181 caatttcaca cagaattcat taaagaggag aaattaacta tgagaggatc gcatcaccat
      241 caccatcacg gatccgctct tgaagtcctc tttcagggac ccgccaagat ggcatttacg
      301 cttgctgacc gcgtgacgga ggagatgctt gccgacaagg cggcgcttgt cgttgaagtg
      361 gtcgaggaaa attatcacga tgcgccgatc gtcggcatcg ctgtggtcaa cgaacatgga
      421 cggtttttcc tgcgcccgga gacggcgctt gccgatccgc agtttgtcgc ctggcttggt
      481 gatgaaacga agaaaaaaag catgtttgac tcaaagcgcg cggcagtcgc cttgaaatgg
      541 aaaggaattg agctatgcgg cgtttccttt gatttattgc tggccgccta tttgcttgat
      601 ccggcgcaag gtgttgatga tgtggctgcc gcagcaaaaa tgaagcaata cgaagcggtg
      661 cgcccggatg aagcggtgta tggcaaaggg gcgaagcggg ccgtgccgga tgagccagtg
      721 ctcgccgagc atttggtccg caaggcggcg gcgatttggg aactcgaacg gccgtttttg
      781 gatgagctgc gccgcaacga acaagatcgg ttgctcgtcg agctggagca gccgttgtct
      841 tcgattttgg cggaaatgga atttgccgga gtgaaagtgg atacgaagcg gctcgaacag
      901 atgggcaaag agctcgccga gcagctgggc acggtcgagc agcgcattta tgagctcgcc
      961 ggccaagaat tcaacatcaa ttcaccgaaa cagctcggcg tcattttatt tgaaaaactg
     1021 cagctgcccg tcttgaaaaa aacgaaaacc ggctactcca cttcggcgga tgtgcttgaa
     1081 aaacttgcgc cttatcacga gatcgtggaa aacattttgc attaccgcca gcttggcaag
     1141 ttgcagtcga cgtatattga aggattgctg aaagtcgtgc gacccgcgac aaagaaggtg
     1201 catacgattt tcaatcaggc gttgacgcaa accggacggc tcagctcgac ggagccgaac
     1261 ttgcaaaaca ttccgattcg gcttgaggaa ggacggaaaa tccgccaagc gttcgtgcca
     1321 tcggagtctg attggctcat tttcgccgcc gactactcgc aaattgagtt gcgcgtcctc
     1381 gcccatattg cggaagatga caatttaatg gaagcgttcc gccgcgattt ggatatccat
     1441 acgaaaacag cgatggacat tttccaagtg agcgaggacg aagtgacgcc caacatgcgc
     1501 cgtcaggcga aggcggtcaa ctatgggatc gtttacggga tcagtgatta cggcttggcg
     1561 caaaacttaa atatttcgcg caaagaggcg gctgaattca tcgagcgcta cttcgagagc
     1621 ttccctggcg tgaagcggta tatggaaaac attgtgcaag aagcaaaaca gaaagggtat
     1681 gtgacgacgc tgctgcatcg gcgccgctat ttgccggata tcacgagccg caacttcaac
     1741 gtccgcagct ttgctgaacg gatggcgatg aacacgccga ttcaagggag cgccgctgac
     1801 attattaaaa aggcgatgat cgatctgaac gccagactga aggaagagcg gctgcaagcg
     1861 caccttttgc tgcaggtgca tgacgagctc attttggagg cgccgaaaga agagatggag
     1921 cggctgtgcc ggctcgttcc ggaagtgatg gagcaagcgg tcacacttcg cgtgccgctc
     1981 aaagtcgatt accattatgg ctcgacgtgg tatgatgcga aataaaagct tccggcttat
     2041 cggtcagttt cacctgattt acgtaaaaac ccgcttcggc gggtttttgc ttttggaggg
     2101 gcagaaagat gaatgactgt ccacgacgct atacccaaaa gaaagcggct tatcggtcag
     2161 tttaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     2221 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     2281 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccctg
     2341 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     2401 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     2461 gtcgagatcc cggacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc
     2521 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     2581 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     2641 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     2701 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     2761 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     2821 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     2881 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     2941 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     3001 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     3061 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     3121 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     3181 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     3241 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     3301 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     3361 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     3421 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     3481 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     3541 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     3661 cactcattag gtaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     3721 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     3781 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     3841 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     3901 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     3961 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     4021 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     4081 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     4141 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     4201 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     4261 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     4321 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     4381 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     4441 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     4501 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     4561 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     4621 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4681 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4741 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4801 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4861 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4921 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4981 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt caacccgacc
     5341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5461 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5581 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5641 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5701 cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca
     5761 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc
     5821 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     5881 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     5941 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     6001 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     6061 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     6121 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     6181 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     6241 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     6301 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     6361 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     6421 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     6481 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     6541 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag
     6601 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6661 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     6721 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     6781 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     6841 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     6901 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     6961 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     7021 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     7081 ctgcgcgtaa ccaccacacc c
//
