LOCUS       sequence-352448-        8032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Pfu DNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8032)
  AUTHORS   Goldman S, Kim R, Hung LW, Jancarik J, Kim SH
  TITLE     Purification, crystallization and preliminary X-ray crystallographic
            analysis of Pyrococcus furiosus DNA polymerase.
  JOURNAL   Acta Crystallogr D Biol Crystallogr. 1998 Sep 1. 54(Pt 5):986-8.
  PUBMED    9757114
REFERENCE   2  (bases 1 to 8032)
  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: "Acta 
            Crystallogr D Biol Crystallogr. 1998 Sep 1. 54(Pt 5):986-8."
COMMENT     Sequence Label: pET16B.Pfu
FEATURES             Location/Qualifiers
     source          1..8032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1457..1486
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1499..1510
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3897..3915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3911..3958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4133..4138,4139..4155,4156..4161))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4188..4206)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4274..4378
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4379..4447,4448..5239)
                     /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(4597..4616)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5410..5998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5899..5918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6152..6169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6184..6323
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6249..6268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6409..6431
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6425..6616)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatatacc atgggccatc atcatcatca tcatcatcat catcacagca gcggccatat
     1501 cgaaggtcgt catatgattt tagatgtgga ttacataact gaagaaggaa aacctgttat
     1561 taggctattc aaaaaagaga acggaaaatt taagatagag catgatagaa cttttagacc
     1621 atacatttac gctcttctca gggatgattc aaagattgaa gaagttaaga aaataacggg
     1681 ggaaaggcat ggaaagattg tgagaattgt tgatgtagag aaggttgaga aaaagtttct
     1741 cggcaagcct attaccgtgt ggaaacttta tttggaacat ccccaagatg ttcccactat
     1801 tagagaaaaa gttagagaac atccagcagt tgtggacatc ttcgaatacg atattccatt
     1861 tgcaaagaga tacctcatcg acaaaggcct aataccaatg gagggggaag aagagctaaa
     1921 gattcttgcc ttcgatatag aaaccctcta tcacgaagga gaagagtttg gaaaaggccc
     1981 aattataatg attagttatg cagatgaaaa tgaagcaaag gtgattactt ggaaaaacat
     2041 agatcttcca tacgttgagg ttgtatcaag cgagagagag atgataaaga gatttctcag
     2101 gattatcagg gagaaggatc ctgacattat agttacttat aatggagact cattcgactt
     2161 cccacattta gcgaaaaggg cagaaaaact tgggattaaa ttaaccattg gaagagatgg
     2221 aagcgagccc aagatgcaga gaataggcga tatgacggct gtagaagtca agggaagaat
     2281 acatttcgac ttgtatcatg taataacaag gacaataaat ctcccaacat acacactaga
     2341 ggctgtatat gaagcaattt ttggaaagcc aaaggagaag gtatacgccg acgagatagc
     2401 aaaagcctgg gaaagtggag agaaccttga gagagttgcc aaatactcga tggaagatgc
     2461 aaaggcaact tatgaactcg ggaaagaatt ccttccaatg gaaattcagc tttcaagatt
     2521 agttggacaa cctttatggg atgtttcaag gtcaagcaca gggaaccttg tagagtggtt
     2581 cttacttagg aaagcctacg aaagaaacga agtagctcca aacaagccaa gtgaagagga
     2641 gtatcaaaga aggctcaggg agagctacac aggtggattc gttaaagagc cagaaaaggg
     2701 gttgtgggaa aacatagtat acctagattt tagagcccta tatccctcga ttataattac
     2761 ccacaatgtt tctcccgata ctctaaatct tgagggatgc aagaactatg atatcgctcc
     2821 tcaagtaggc cacaagttct gcaaggacat ccctggtttt ataccaagtc tcttgggaca
     2881 tttgttagag gaaagacgaa agattaagac aaaaatgaag gaaactcaag atcctataga
     2941 aaaaatactc cttgactata gacaaaaagc gataaaactc ttagcaaatt ctttctacgg
     3001 atattatggc tatgcaaaag caagatggta ctgtaaggag tgtgctgaga gcgttactgc
     3061 ctggggaaga aagtacatcg agttagtatg gaaggagctc gaagaaaagc ttggatttaa
     3121 agtcctctac attgacactg atggtctcta tgcaactatc ccaggaggag aaagtgagga
     3181 aataaagata aaggctctag aatttgtaaa atacataaat tcaaagctcc ctggactgct
     3241 agagcttgaa tatgaagggt tttataagag gggattcttc gttacgaaga agaggtatgc
     3301 agtaatagat gaagaaggaa aagtcattac tcgtggttta gagatagtta ggagagattg
     3361 gagtgaaatt gcaaaagaaa ctcaagctag agttttggag acaatactaa aacacggaga
     3421 tgttgaagaa gctgtgagaa tagtaaaaga agtaatacaa aagcttgcca attatgaaat
     3481 tccaccagag aagctcgcaa tatatgagca gataacaaga ccattacatg agtataaggc
     3541 gataggtcct cacgtagctg ctgcaaagaa actagctgct aaaggagtta aaataaagcc
     3601 aggaatggta attggataca tagtacttag aggcgatggt ccaattagca atagggcaat
     3661 tctagctgag gaatacgatc ccaaaaagca caagtatgac gcagaatatt acattgagaa
     3721 ccaggttctt ccagcggtac ttaggatatt ggagggattt ggatacagaa aggaagacct
     3781 cagataccaa aagacaagac aagtcggcct aacttcctgg cttaacatta aaaaatccta
     3841 gctcgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     3901 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3961 gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag
     4021 cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat
     4081 ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc
     4141 ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg
     4201 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4261 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4321 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4381 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4441 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4501 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4561 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4621 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4681 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4741 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4801 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4861 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4921 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4981 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5041 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5101 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5161 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5221 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5281 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5341 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5401 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5461 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5521 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     5581 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5641 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5701 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5761 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5821 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5881 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5941 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6001 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6061 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6121 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6181 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     6241 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     6301 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     6361 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     6421 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     6481 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     6541 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     6601 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     6661 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     6721 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     6781 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     6841 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     6901 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     6961 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     7021 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     7081 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     7141 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     7201 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     7261 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     7321 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     7381 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     7441 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     7501 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     7561 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     7621 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     7681 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     7741 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     7801 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     7861 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     7921 gttgcgctca ctgcccgctt tccagtcggg aaactgtcgt gccagctgca ttaatgaatc
     7981 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt tt
//
