LOCUS       sequence-438176-        6150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For recombinant expression of vanB (PP_3737 from Pseudomonas putida)
            with the A24P mutation and an N-terminal thrombin-cleavable His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  AUTHORS   Bleem AC, Kuatsjah E, Johnsen J, Mohamed ET, Alexander WG, 
            Kellermyer ZA, Carroll AL, Rossi R, Schlander IB, Peabody V GL, Guss
            AM, Feist AM, Beckham GT
  TITLE     Evolution and engineering of pathways for aromatic O-demethylation 
            in Pseudomonas putida KT2440.
  JOURNAL   Metab Eng. 2024 Jul;84:145-157. doi: 10.1016/j.ymben.2024.06.009. 
            Epub 2024 Jun 25.
  PUBMED    38936762
REFERENCE   2  (bases 1 to 6150)
  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: "Metab 
            Eng."; date: "2024-07"; volume: "84:145-157. doi"; pages: " 
            10.1016/j.ymben.2024.06.009. Epub 2024 Jun 25"
COMMENT     Sequence Label: pACB143
FEATURES             Location/Qualifiers
     source          1..6150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(90..109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     300..321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(550..1365)
                     /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             complement(550..1365)
                     /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     1273..1292
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1357..1376
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1487..2075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1976..1995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2229..2246
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2261..2400
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2326..2345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2486..2508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2502..2693)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3502..4584)
                     /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     4546..4565
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4585..4662)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4868..4887
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4971..4990
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4971..4989
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4990..5014
                     /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             5029..5051
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5070..5087
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5097..5114
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      6069..6116
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
       61 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      121 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      181 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      241 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      301 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      361 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
      421 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
      481 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
      541 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
      601 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
      661 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
      721 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
      781 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
      841 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
      901 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
      961 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     1021 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     1081 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     1141 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     1201 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     1261 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     1321 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     1381 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     1441 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1501 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1561 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1621 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1681 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1741 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1801 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     1861 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     1921 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     1981 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2041 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2101 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2161 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2221 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     2281 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     2341 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     2401 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     2461 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2521 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     2581 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     2641 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     2701 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     2761 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     2821 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     2881 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     2941 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3001 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3061 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3121 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     3181 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     3241 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     3301 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     3361 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     3421 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     3481 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3541 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     3601 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     3661 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     3721 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     3781 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     3841 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     3901 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     3961 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4021 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4081 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     4141 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     4201 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     4261 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     4321 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     4381 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     4441 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     4501 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     4561 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     4621 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     4681 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     4741 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     4801 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     4861 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     4921 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     4981 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     5041 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg
     5101 tgccgcgcgg cagccatatg atcgatgccg tagtggtatc ccgtaacgat gaagcgcagg
     5161 gtatctgcag cttcgagctg gccgcgccag atggcagcct gctgccggcg ttcagcgccg
     5221 gcgcccatat cgacgtgcac ctgcccgacg ggctggtgcg ccagtattcg ctgtgcaacc
     5281 accccgaaga acgccatcgc tatctgattg gcgtactcaa cgacccggct tcgcggggcg
     5341 gttctcgtag cctgcacgaa caggtgcaag ccggtgcccg gctgcgtatc agtgcgccgc
     5401 gcaacctgtt cccgctggcc gagggtgcgc agcgcagttt gctgtttgct ggcggtatcg
     5461 gcattacccc aatcctgtgc atggccgagc agctgtccga cagcggccag gccttcgagc
     5521 tgcactactg tgcccgctcc agcgagcgtg cggcgtttgt cgagcggatc cgcagcgcgc
     5581 cgttcgctga tcggctgttc gtgcattttg acgagcagcc ggaaacggcg ctggacatcg
     5641 cccaggtgct gggcaacccg caagatgatg tgcacctgta tgtatgcggg cccggcgggt
     5701 tcatgcagca tgtgctggac agcgcgaagg ggctgggctg gcaggaggcc aacctgcacc
     5761 gcgagtactt cgccgcagca ccggtggatg ccagcaacga tggcagtttc gcggtgcagg
     5821 tgggcagcac gggacaggtg ttcgaggtgc cagccgaccg gaccgtggtg caggtgctgg
     5881 aagagaatgg tatcgagatc gccatgtcgt gcgagcaggg tatttgcggc acctgcctga
     5941 cacgcgtgct gcagggcaca ccggaccatc gcgatctgtt tctcaccgaa gaggaacagg
     6001 ccctgaacga tcagttcacg ccctgctgct cgcgctcgaa gacgccgctg ctggtgctgg
     6061 acatctgact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     6121 aaggaggaac tatatccgga ttagcgaatg
//
