LOCUS       sequence-318950-        5910 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  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 5910)
  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..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..2758
                     /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(2773..2790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2944..3532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3024..3043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3643..3662)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3654..4469
                     /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             3654..4469
                     /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(3727..3746)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4561..5016)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4698..4719)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4910..4929
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5053..5100)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5096..5114
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5167..5184)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5815..5832)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5842..5859)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5878..5900
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
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 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     2761 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2821 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2881 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2941 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3001 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3061 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3121 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3181 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3241 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3301 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3361 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3421 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3481 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3541 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3601 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     3661 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     3721 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     3781 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     3841 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     3901 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     3961 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4021 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4081 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4141 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4201 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4261 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     4321 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     4381 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     4441 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     4501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4561 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4621 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4681 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4741 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     4801 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     4861 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4921 cgaaaggagc gggcgctagg gcgttggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4981 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt
     5041 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
     5101 ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg
     5161 atctcagtgg tggtggtggt ggtgctcgag ttattctttt ttacctgcct gacggaatgt
     5221 caggttgtag cggcagtcga tggtgagtgg atgaaacccc gctttcaacg gttgaatacc
     5281 gtgataaaac agccgcgatt caccgcccca taccaccaca tcgccatgtt ccaacaacaa
     5341 acgtttgagc ggatcattac gtttcaggcc gccaaattga aaaatcgcgg gtaagcccag
     5401 agaaacagaa acaattggcg cgcgcagatc cggttcggtt ttatcctgat gcagcgacag
     5461 tttcgcgcca ggagcgtagc ggttgataag acaagcatct ggctggaaat ctggatagcc
     5521 cgccgccgta gccgcacgtt gacataaatt atgaaaactc tgtggcatgg cgggccacgg
     5581 tttatttgtt tgcggatcaa tgggcgaata gagataacct tgccgatggg tcgtccagcc
     5641 cagatgccca cagttggtca tcgccaccga catggtatat cccccggggg tgaccatctg
     5701 gcgaaacggc gactggctgg caacgtcatt aatatcgcgg attagttgct ccgcagcgtt
     5761 aaaagcaaaa cgccgtaaaa ttaccgctcc agccgccagt ggctcttgca tatggctgcc
     5821 gcgcggcacc aggccgctgc tgtgatgatg atgatgatgg ctgctgccca tggtatatct
     5881 ccttcttaaa gttaaacaaa attatttcta
//
