LOCUS       sequence-392530-        8224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PfuX7 DNA polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8224)
  AUTHORS   Norholm MH
  TITLE     A mutant Pfu DNA polymerase designed for advanced uracil-excision 
            DNA engineering.
  JOURNAL   BMC Biotechnol. 2010 Mar 16;10:21. doi: 10.1186/1472-6750-10-21.
  PUBMED    20233396
REFERENCE   2  (bases 1 to 8224)
  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: "BMC 
            Biotechnol."; date: "2010-03-16"; volume: "10:21. doi"; pages: " 
            10.1186/1472-6750-10-21"
COMMENT     Sequence Label: pET-PfuX7
FEATURES             Location/Qualifiers
     source          1..8224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2888..2905)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2915..2937
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2952..2976
                     /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     complement(2976..2995)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2977..2995)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3079..3098)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3304..3381
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(3401..3420)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5775..5966
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5960..5982)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6068..6207
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6123..6142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6222..6239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6393..6981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6473..6492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7152..7943,7944..8012))
                     /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     7775..7794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8013..8117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8185..8203
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atctcaaaca gatgcacgac gaattgctac tttttctgct
      361 tctccagcat ctgcagcagc tccttcggcg cgtccttttc gcttaccgca ccacggccgg
      421 tcttgccacc gccctcgtcg taggtgaagg agatcatctt gcccacacgc catactttct
      481 tgatcttgga gatgtctacc tctttttctt cgcctttgta cttgaacttt acggttgcac
      541 cgccaccgcc ggtaccggat tttttaatgt taagccagga agttaggccg acttgtcttg
      601 tcttttggta tctgaggtct tcctttctgt atccaaatcc ctccaatatc ctaagtaccg
      661 ctggaagaac ctggttctca atgtaatatt ctgcgtcata cttgtgcttt ttgggatcgt
      721 attcctcagc tagaattgcc ctattgctaa ttggaccatc gcctctaagt actatgtatc
      781 caattaccat tcctggcttt attttaactc ctttagcagc tagtttcttt gcaacagcta
      841 cgtgaggacc tatcgcctta tactcatgta atggtcttgt tatctgctca tatattgcga
      901 gcttctctgg tggaatttca taattggcaa gcttttgtat tacttctttt actattctca
      961 cagcttcttc aacatctccg tgttttagta ttgtctccaa aactctagct tgagtttctt
     1021 ttgcaatttc actccaatct ctcctaacta tctctaaacc acgagtaatg acttttcctt
     1081 cttcatctat tactgcatac ctcttcttcg taacgaagaa tcccctctta taaaaccctt
     1141 catattcaag ctctagcagt ccagggagct ttgaatttat gtattttaca aattctagag
     1201 cctttttctt tatttcctca ctttctcctc ctgggatagt tgcatagaga ccatcagtgt
     1261 caatgtagag gactttaaat ccaaactttt cttcgagctc cttccatact aactcgatgt
     1321 actttcttcc ccaggcagta acgctctcag cacactcctt acagtaccat cttgcttttg
     1381 catagccata atatccgtag aaagaatttg ctaagagttt tatcgctttt tgtctatagt
     1441 caaggagtat tttttctata ggatcttgag tttccttcat ttttgtctta atcttttgtc
     1501 tttcctctaa caaatgtccc aagagacttg gtataaaacc agggatgtcc ttgcagaact
     1561 tgtggcctac ttgaggagcg atatcatagt tcttgcatcc ctcaagattt agagtatcgg
     1621 gagaaacatt gtgggtaatt ataatcgagg gatatagggc tctaaaatct aggtatacta
     1681 tgttttccca caaccccttt tctggctctt taacgaatcc acctgtgtag ctctccctga
     1741 gccttctttg atactcctct tcacttggct tgtttggagc tacttcgttt ctttcgtagg
     1801 ctttcctaag taagaaccac tctacaaggt tccctgtgct tgaccttgaa acatcccata
     1861 aaggttgtcc aactaatctt gaaagctgaa tttccattgg aaggaattct ttcccgagtt
     1921 cataagttgc ctttgcatct tccatcgagt atttggcaac tctctcaagg ttctctccac
     1981 tttcccaggc ttttgctatc tcgtcggcgt ataccttctc ctttggcttt ccaaaaattg
     2041 cttcatatac agcctctagt gtgtatgttg ggagatttat tgtccttgtt attacatgat
     2101 acaagtcgaa atgtattctt cccttgactt ctacagccgt catatcgcct attctctgca
     2161 tcttgggctc gcttccatct cttccaatgg ttaatttaat cccaagtttt tctgcccttt
     2221 tcgctaaata tgggaagtcg aatgagtctc cattataagt aactataatg tcaggatcct
     2281 tctccctgat aatcctgaga aatctcttta tcatctctct ctcgcttgat acaacctcaa
     2341 cgtatggaag atctatgttt ttccaagtaa tcacctttgc ttcattttca tctgcataac
     2401 taatcattat aattgggcct tttccaaact cttctccttc gtgatagagg gtttctatat
     2461 cgaaggcaag aatctttagc tcttcttccc cctccattgg tattaggcct ttgtcgatga
     2521 ggtatctctt tgcaaatgga atatcgtatt cgaagatgtc cacaactgct ggatgttctc
     2581 taactttttc tctaatagtg ggttgatctt ggggatgttc caaataaagt ttccacacgg
     2641 taataggctt gccgagaaac tttttctcaa ccttctctac atcaacaatt ctcacaatct
     2701 ttccatgcct ttcccccgtt attttcttaa cttcttcaat ctttgaatca tccctgagaa
     2761 gagcgtaaat gtatggtcta aaagttctat catgctctat cttaaatttt ccgttctctt
     2821 ttttgaatag cctaataaca ggttttcctt cttcagttat gtaatccaca tctaaaatca
     2881 ttgatccgtg atggtgatgg tgatgcatgg tatatctcct tcttaaagtt aaacaaaatt
     2941 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     3001 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     3061 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     3121 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     3181 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     3241 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     3301 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     3361 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     3421 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     3481 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     3541 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     3601 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     3661 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     3721 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     3781 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     3841 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     3901 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     3961 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     4021 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     4081 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     4141 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     4201 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     4261 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     4321 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     4381 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacagttt
     4441 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     4501 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     4561 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     4621 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     4681 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     4741 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     4801 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
     4861 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
     4921 aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc
     4981 ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca
     5041 tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg
     5101 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
     5161 acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc
     5221 aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc
     5281 gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga
     5341 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     5401 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     5461 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     5521 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     5581 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     5641 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     5701 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     5761 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     5821 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     5881 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     5941 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6001 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6061 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     6121 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga
     6181 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     6241 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6301 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6361 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6421 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6481 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6541 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6601 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6661 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6721 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6781 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6841 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6901 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6961 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     7021 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     7081 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7141 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7201 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7261 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7321 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7381 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7441 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     7501 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7561 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7621 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7681 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7741 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact
     7801 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7861 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7921 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7981 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     8041 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     8101 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     8161 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
     8221 ttaa
//
