LOCUS       sequence-361774-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Br512_Mut235 polymerase in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  AUTHORS   Paik I, Ngo PHT, Shroff R, Diaz DJ, Maranhao AC, Walker DJF, Bhadra 
            S, Ellington AD
  TITLE     Improved Bst DNA Polymerase Variants Derived via a Machine Learning 
            Approach.
  JOURNAL   Biochemistry. 2021 Nov 11. doi: 10.1021/acs.biochem.1c00451.
  PUBMED    34762799
REFERENCE   2  (bases 1 to 6218)
  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: 
            "Biochemistry. 2021 Nov 11. doi: 10.1021/acs.biochem.1c00451."
COMMENT     Sequence Label: pKAR2-Br512_Mut235
FEATURES             Location/Qualifiers
     source          1..6218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      16..62
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(359..985)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     890..909
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(join(1014..1805,1806..1874))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1637..1656
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1875..1946)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      2023..2109
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2253..2270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2424..3012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2504..2523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3822..3841
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3906..3925
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4069..4087
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    4108..4126
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     primer_bind     4127..4146
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4127..4145
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4147..4165
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        4185..4234
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             4268..4276
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 atcctaagct taatttagca taaccccttg gggcctctaa acgggtcttg aggggttttt
       61 tgagctgagc ttggactcct gttgatagat ccggccggta atgacctcag aactccatct
      121 gcctaatgga gtgattctag ccggtcgtta cacgtggaac gggaactgcc agacatcaaa
      181 taaaacaaaa ggctcagtcg gaagactggg ccttttgttt tatctgttgt ttgtcggtga
      241 acactctccc ggaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      301 tcacctagat ccttttaaac tagtgaagtt accatcacgg aaaaaggtta tgctgctttt
      361 aagacccact ttcacattta agttgttttt ctaatccgca aatgatcaat tcaaggccga
      421 ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt cgtaataatg
      481 gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga tgctcttgat
      541 cttccaatac gcaacctaaa gtaaaatgcc ccactgcact gagtgcatat aatgcattct
      601 ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttcgagagtt tcatactgtt
      661 tttctgtagg ccgtgtacct aaatgtactt ttgctccatc gcgatgactt agtaaagcac
      721 atctaaaact tttagcgtta ttacgtaaaa aatcttgcca gctttcccct tctaaagggc
      781 aaaagtgagt atggtgccta tctaacatct caatggctaa ggcgtcgagc aaagcccgct
      841 tattttttac atgccaatac aatgtaggct gctctacacc tagcttctgg gcgagtttac
      901 gggttgttaa accttcgatt ccgacctcat taagcagctc taatgcgctg ttaatcactt
      961 tacttttatc taaacgagac atcattaacc tcctcaagag gatcgaatag ttattaccaa
     1021 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1081 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1141 gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1201 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatc
     1261 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1321 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1381 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1441 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1501 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1561 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1621 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1681 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1741 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1801 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1861 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1921 ctcatgagcg gatacatatt tgaatgtatt tagaaaaatt ttttaaggca gttattggtg
     1981 ccgcttaaac gcctggggta atgactctct agcttgaggc atcaaataaa acgaaaggct
     2041 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2101 aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa cggaatttcg
     2161 ggcagcgttg ggtcctggcc acgggtgcgc cggtgtgaaa taccgcacag atgcgtaagg
     2221 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2281 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2341 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2401 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2461 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2521 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2581 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2641 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2701 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2761 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2821 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     2881 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2941 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3001 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3061 aactcacgtt aagggatttt ggtcatggaa ttaattctta gaaaaactca tcgagcatca
     3121 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     3181 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     3241 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     3301 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     3361 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     3421 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaag acgaaatacg
     3481 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     3541 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     3601 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     3661 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     3721 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     3781 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     3841 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     3901 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtg
     3961 taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggggag
     4021 tttagggaaa gagcatttgt catcatgacc atgacattaa cctataaaaa taggcgtatc
     4081 acgaggccct ttccctaggg tcttcacact ctatcattga tagagttaat acgactcact
     4141 atagggtccc tatcagtgat agagagaatt cgtactgagc acagctgtca ccggatgtgc
     4201 tttccggtct gatgagtccg tgaggacgaa acagcctcta caaataattt tgtttaaact
     4261 agttagataa ggaggttaca tatgcaccat catcacggtc atcaccacca tccgcgtggt
     4321 gttgacccga gccgtaagga gaaccacctg tctgacgaag acttcaaggc ggtgttcggt
     4381 atgacccgtt ctgcgttcgc gaacctgccg ctgtggaaac aacagaacct gaagaaggag
     4441 aaaggtctgt tcggttctgg aagcgcagca gcacctaaga tggcattcac attggccgat
     4501 cgtgtcaccg aagagatgct ggcagacaag gcagccttgg tcgtggaggt agttgaggag
     4561 aactatcacg acgcaccgat tgttggaatc gccgtggtca atgaacatgg tcgcttcttc
     4621 ttgcgccctg agactgcgtt ggccgaccca caattcgtgg cctggttagg agatgaaacg
     4681 aagaagaagt caatgttcga cagcaaacgc gcagccgtag ctctgaagtg gaaaggaatt
     4741 gagctgtgtg gtgtgagttt cgaccttctc ttagcagcgt acttgcttga tcccgctcaa
     4801 ggcgtcgacg acgtggcagc cgctgccaag atgaagcaat atgaagcggt gcgtccggat
     4861 gaggctgtgt acgggaaggg agctaaacgc gcggtgcctg atgaacccgt gcttgctgag
     4921 cacttggtac gcaaggctgc ggctatctgg gagctggagc gtcccttcct ggatgagttg
     4981 cgtcgcaacg agcaggaccg cctgcttgta gagttagaac agcctcttag ctctattctt
     5041 gccgagatgg agttcgctgg tgtcaaagta gataccaagc gccttgagca aatgggtaag
     5101 gagttggctg aacaactggg cacagtggaa cagcgtatct acgaactggc cggtcaggag
     5161 ttcaacatca acagccccaa gcagctggga gtgatcctgt tcgagaagtt gcagctgcca
     5221 gtattgaaga agactaagac tggctacagt acctcggctg acgtactgga gaagctggct
     5281 ccttaccatg agatcgtgga gaacatcttg cactaccgcc agctgggcaa gctgcagtct
     5341 acctacattg agggtctgtt aaaggtcgtg cgtccagaca cgaagaaggt gcatacgatc
     5401 ttcaatcagg cgctgaccca aactggtcgt ttgtcgtcca cagagcccaa tcttcagaat
     5461 atccctattc gtcttgagga aggccgcaag attcgccagg ccttcgttcc ttcggaatcg
     5521 gactggctga tcttcgcagc agattacgat cagatcgagc ttcgcgtgtt ggcacatatc
     5581 gcggaggatg acaacttaat ggaggcgttc cgccgcgatc tggatatcca tactaagacc
     5641 gcgatggata tcttccaagt gtcagaagac gaggtaacac cgaacatgcg acgccaggcg
     5701 aaagcggtta acttcggcat cgtctacggc atcagcgact atggcctggc ccagaacttg
     5761 aacatcagcc gcaaggaggc agccgagttc atcgagcgct acttcgagag tttcccaggt
     5821 gtgaagcgtt atatggagaa tatcgtacaa gaggcgaagc agaaaggcta cgtgaccacg
     5881 ctgttacacc gtcgtcgcta ccttcctgat atcaacagcc gtaacttcaa tgtacgttcc
     5941 ttcgccgaac gcatggcgat gaataccccc atccaggggt cagctgcaga tatcatcaag
     6001 aaagctatga tcgacttaaa cgctcgtctg aaggaagaac gcttacaggc gcacctctta
     6061 ctgcaagtcc atgacgaatt gatccttgag ggccccaagg aagagatgga gcgtctttgc
     6121 cgtctggtgc cggaagtaat ggaacaggcc gtcacgctgc gcgtacctct gaaagtcgat
     6181 taccactacg gctccacctg gtatgacgcc aagtaagg
//
