LOCUS       sequence-311016-        5031 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  A plasmid for expression in E. coli to produce His-tag fusion 
            protein AlkB with Asp at position 135 mutated to Thr.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5031)
  AUTHORS   Wang Y, Katanski CD, Watkins C, Pan JN, Dai Q, Jiang Z, Pan T
  TITLE     A high-throughput screening method for evolving a demethylase enzyme
            with improved and new functionalities.
  JOURNAL   Nucleic Acids Res. 2021 Mar 18;49(5):e30. doi: 10.1093/nar/gkaa1213.
  PUBMED    33337498
REFERENCE   2  (bases 1 to 5031)
  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: "2021-03-18"; volume: "49(5):e30. doi"; pages: " 
            10.1093/nar/gkaa1213"
COMMENT     Sequence Label: AlkB-D135T
FEATURES             Location/Qualifiers
     source          1..5031
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    5..29
                     /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(29..48)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(30..48)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(132..151)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        357..434
                     /gene="lacI"
                     /label=lacI promoter
     CDS             435..1517
                     /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(454..473)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2326..2517
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2511..2533)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2619..2761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2674..2693
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2776..2793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2947..3535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3027..3046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3646..3665)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3657..4472
                     /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             3657..4472
                     /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(3730..3749)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4565..5020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4702..4723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4914..4933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
       61 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
      121 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      181 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
      241 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
      301 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
      361 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
      421 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
      481 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
      541 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
      601 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
      661 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
      721 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
      781 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
      841 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
      901 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
      961 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     1021 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     1081 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     1141 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     1201 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     1261 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     1321 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     1381 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     1441 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     1501 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     1561 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     1621 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     1681 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     1741 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     1801 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     1861 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     1921 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     1981 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     2041 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     2101 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     2161 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     2221 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     2281 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     2341 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     2401 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     2461 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2521 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2581 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     2641 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     2701 tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     2761 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2821 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2881 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2941 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3001 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3061 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3121 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3181 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3241 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3301 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3361 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     3421 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3481 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3541 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3601 tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     3661 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg
     3721 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     3781 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     3841 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     3901 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     3961 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     4021 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     4081 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     4141 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     4201 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     4261 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     4321 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     4381 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     4441 agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt
     4501 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4561 cctgaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4621 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     4681 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4741 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4801 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     4861 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     4921 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     4981 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
