LOCUS       sequence-327584-        7824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To produce a dual affinity-tagged Phi29 DNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7824)
  AUTHORS   Blainey PC, Quake SR
  TITLE     Digital MDA for enumeration of total nucleic acid contamination.
  JOURNAL   Nucleic Acids Res. 2011 Mar;39(4):e19. doi: 10.1093/nar/gkq1074. 
            Epub 2010 Nov 11.
  PUBMED    21071419
REFERENCE   2  (bases 1 to 7824)
  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: "Nucleic 
            Acids Res."; date: "2011-03"; volume: "39(4):e19. doi"; pages: " 
            10.1093/nar/gkq1074. Epub 2010 Nov 11"
COMMENT     Sequence Label: pGS-21a his6-GST-WTPhi29DNAP
FEATURES             Location/Qualifiers
     source          1..7824
                     /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             89..742
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             779..793
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2552..2569
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2622..2640)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2636..2683
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2720..3175
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2807..2826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3017..3038
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3201..3305
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3306..3374,3375..4166)
                     /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     complement(3524..3543)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4337..4925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4826..4845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5079..5096
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5111..5250
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5176..5195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5336..5358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5352..5543)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6352..7434)
                     /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     7396..7415
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7435..7512)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7718..7737
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gtcccctata ctaggttatt ggaaaattaa
      121 gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca
      181 tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga
      241 gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat
      301 catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga
      361 gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata
      421 tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa
      481 aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc
      541 tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga
      601 tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa
      661 gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg
      721 tggtggcgac catcctccaa aatcggatct gggccacaca ggccatagat ctggtaccga
      781 cgacgacgac aaggccatgg ctgatatcgg atccatgccg cgtaaaatgt attcctgtga
      841 ttttgaaacg acgacgaaag ttgaagattg ccgtgtgtgg gcgtatggtt atatgaacat
      901 cgaagaccat tcagaatata aaattggcaa ctcgctggat gaatttatgg cgtgggtgct
      961 gaaagttcag gccgacctgt acttccacaa tctgaaattt gatggtgcgt tcattatcaa
     1021 ctggctggaa cgtaatggct ttaaatggag cgccgatggt ctgccgaaca cctataatac
     1081 gattatctct cgtatgggcc aatggtatat gattgatatc tgcctgggct acaaaggtaa
     1141 acgcaaaatt cataccgtga tctatgacag cctgaaaaaa ctgccgtttc cggtgaagaa
     1201 aattgcgaaa gatttcaaac tgaccgtcct gaaaggcgat attgactatc acaaagaacg
     1261 tccggttggt tacaaaatca cgccggaaga atatgcgtac attaaaaacg atatccagat
     1321 tatcgcagaa gctctgctga ttcagtttaa acaaggcctg gatcgcatga ccgccggcag
     1381 tgactccctg aaaggtttca aagatatcat caccacgaaa aaattcaaaa aagtgttccc
     1441 gaccctgagc ctgggtctgg ataaagaagt tcgttatgca taccgcggcg gttttacgtg
     1501 gctgaacgac cgtttcaaag aaaaagaaat tggcgagggt atggtctttg atgtgaatag
     1561 tctgtatccg gctcagatgt actcccgcct gctgccgtat ggcgaaccga tcgttttcga
     1621 gggtaaatat gtctgggatg aagactaccc gctgcatatt cagcacatcc gttgtgaatt
     1681 tgaactgaaa gaaggctata ttccgaccat tcaaatcaaa cgtagccgct tctataaggg
     1741 taacgaatac ctgaaaagct ctggcggtga aatcgcagac ctgtggctga gtaacgtcga
     1801 tctggaactg atgaaagaac attacgatct gtacaacgtt gaatacatct ccggcctgaa
     1861 atttaaagcc accacgggtc tgtttaaaga tttcattgac aaatggacct acatcaaaac
     1921 cacgtctgaa ggtgcaatca aacagctggc taaactgatg ctgaacagcc tgtatggcaa
     1981 atttgcatct aatccggatg ttaccggtaa agtcccgtac ctgaaagaaa atggcgctct
     2041 gggttttcgc ctgggcgaag aagaaaccaa agatccggtg tatacgccga tgggtgtttt
     2101 cattaccgcg tgggcacgtt acaccacgat cacggcagca caagcgtgct atgaccgcat
     2161 tatctactgt gataccgact caattcatct gaccggcacg gaaatcccgg atgtgattaa
     2221 agatatcgtt gacccgaaaa aactgggtta ttgggcacac gaatcgacct ttaaacgtgc
     2281 taaatacctg cgccagaaaa cgtacatcca agacatctac atgaaagaag tcgatggcaa
     2341 actggtggaa ggttcaccgg atgactatac cgacattaaa ttttcggtga aatgcgccgg
     2401 catgaccgat aaaatcaaaa aagaagtcac gttcgaaaat ttcaaagtgg gtttcagtcg
     2461 caaaatgaaa ccgaaaccgg tgcaagttcc gggcggcgtt gtcctggtgg atgatacctt
     2521 cacgattaaa cagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc
     2581 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     2641 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     2701 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2761 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2821 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2881 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2941 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3001 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3061 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3121 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     3181 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3241 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3301 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3361 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3421 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3481 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3541 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3601 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3661 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3721 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3781 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3841 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3901 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3961 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4021 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4081 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4141 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4201 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4261 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4321 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4381 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4441 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4501 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4561 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4621 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4681 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4741 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4801 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4861 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4921 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4981 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5041 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5101 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     5161 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     5221 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     5401 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     5461 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     5521 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5581 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5641 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5701 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5761 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5821 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5881 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5941 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     6001 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     6061 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     6121 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     6181 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     6241 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6301 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6361 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6421 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6481 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6541 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6601 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     6661 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6721 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6781 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6841 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6901 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6961 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     7021 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     7081 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     7141 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     7201 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     7261 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     7321 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     7381 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7441 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7501 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     7561 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     7621 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7681 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7741 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     7801 agatcgatct cgatcccgcg aaat
//
