LOCUS       sequence-443111-        6207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For recombinant expression of fghA (PP_1617 from Pseudomonas putida)
            with five mutations (K10N, S11R, W15C, G76V, G258D) and a C-terminal
            His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6207)
  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 6207)
  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: pACB120
FEATURES             Location/Qualifiers
     source          1..6207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(71..89)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      85..132
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      169..624
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(256..275)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     466..487
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        650..754
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(755..823,824..1615)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(973..992)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1786..2374
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2275..2294
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2528..2545
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2560..2699
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2625..2644)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2785..2807
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2801..2992)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3801..4883)
                     /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     4845..4864
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4884..4961)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5167..5186
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5270..5289
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5270..5288
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5289..5313
                     /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             5328..5350
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
       61 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      121 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      181 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      241 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      301 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      361 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      421 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      481 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      541 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      601 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      661 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      721 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      781 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
      841 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
      901 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
      961 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1021 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1081 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1141 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1201 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1261 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     1321 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1381 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1441 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1501 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1561 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1621 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1681 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1741 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1801 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1861 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1921 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1981 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2041 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2101 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2161 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2221 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2281 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2341 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2401 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2461 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2521 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     2581 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     2641 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     2701 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     2761 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     2821 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     2881 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     2941 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3001 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3061 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3121 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3181 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     3241 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     3301 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     3361 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     3421 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     3481 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     3541 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     3601 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     3661 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     3721 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     3781 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3841 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     3901 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     3961 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4021 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4081 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4141 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4201 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4261 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     4321 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     4381 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     4441 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     4501 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     4561 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     4621 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     4681 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     4741 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     4801 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     4861 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     4921 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     4981 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5041 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5101 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5161 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     5221 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     5281 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     5341 aagaaggaga tatacatgag cctggataac atctcctgcc agaatagatt cggcggctgc
     5401 cacaagcgtt accggcatca ttccaaggtg ctgggttgcg atatggtgtt cgctgtgtac
     5461 ctgccgccgc aggccgagca gggcgaaaag ctgccggtgc tgtactggct cagcggcctc
     5521 acctgcaccg acgagaactt catgcagaag gccggcgccc agcgtctggc cgccgagctg
     5581 gtgctgatca tcgtcgcccc cgataccagc ccgcgtggcg agcaggtgcc gggcgatccg
     5641 gacggcgcct gggacttcgg cctgggcgct ggcttctacc tcaacgccac ccagcaaccc
     5701 tgggcccagc actaccgcat gcatgactat gtggtggagg agttgccagc cctgatcgag
     5761 gcacacttcc cggcgtcggg cgagcgcagc atcagcggcc actccatggg tgggcatggc
     5821 gcgctggtgt gcgccttgcg caatccgggg cgctaccgct cggtgtcggc attctcgccg
     5881 atcagcaatc cgatggattg tccgtggggc gagaaggcct tcagccgcta cctgggtgaa
     5941 gaccgtgggc gctggcgtga gtgggatgcc agcgtgctgc tggccgaaac cccggcaggc
     6001 caatgcccgc cgttgctggt ggatcagggc gaccgtgacg acttcctcga gaagcaactc
     6061 aagcctgaag cgctggaaca ggcggcacgc aaaggtggcc atgcaatgac cctgcgtatg
     6121 cagccagact atgaccatag ctactacttc attgccagct tcatcgagga gcacctgcgc
     6181 catcatgcgg tggcactggg gcgggtt
//
