LOCUS       sequence-437368-        6207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For recombinant expression of fghA (PP_1617 from Pseudomonas putida)
            with 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: pACB108
FEATURES             Location/Qualifiers
     source          1..6207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             19..36
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(89..107)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      103..150
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      187..642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(274..293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     484..505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        668..772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(773..841,842..1633)
                     /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(991..1010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1804..2392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2293..2312
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2546..2563
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2578..2717
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2643..2662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2803..2825
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2819..3010)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3819..4901)
                     /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     4863..4882
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4902..4979)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5185..5204
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5288..5307
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5288..5306
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5307..5331
                     /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             5346..5368
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gtggcactgg ggcgggttca ccaccaccac caccactgag atccggctgc taacaaagcc
       61 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      121 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      181 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      241 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      361 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      481 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      541 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      601 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
      661 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      721 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      781 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      841 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      901 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      961 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1021 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1081 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1141 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1201 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1261 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     1321 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1381 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1441 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1501 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1561 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1621 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1681 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1741 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1801 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1861 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1921 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1981 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2041 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2101 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2161 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2221 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2281 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2341 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2401 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2461 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2521 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     2581 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     2641 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     2701 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2761 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     2821 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     2881 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     2941 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3001 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3061 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3121 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     3181 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3241 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3301 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3361 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     3421 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     3481 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3541 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3601 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3661 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3721 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3781 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3841 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3901 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3961 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4021 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4081 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4141 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4201 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4261 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4321 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4381 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4441 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4501 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4561 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4621 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4681 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4741 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4801 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4861 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4921 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4981 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5041 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5101 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5161 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5221 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     5281 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     5341 ataattttgt ttaactttaa gaaggagata tacatgagcc tggataacat ctcctgccag
     5401 aagagcttcg gcggctggca caagcgttac cggcatcatt ccaaggtgct gggttgcgat
     5461 atggtgttcg ctgtgtacct gccgccgcag gccgagcagg gcgaaaagct gccggtgctg
     5521 tactggctca gcggcctcac ctgcaccgac gagaacttca tgcagaaggc cggcgcccag
     5581 cgtctggccg ccgagctggg gctgatcatc gtcgcccccg ataccagccc gcgtggcgag
     5641 caggtgccgg gcgatccgga cggcgcctgg gacttcggcc tgggcgctgg cttctacctc
     5701 aacgccaccc agcaaccctg ggcccagcac taccgcatgc atgactatgt ggtggaggag
     5761 ttgccagccc tgatcgaggc acacttcccg gcgtcgggcg agcgcagcat cagcggccac
     5821 tccatgggtg ggcatggcgc gctggtgtgc gccttgcgca atccggggcg ctaccgctcg
     5881 gtgtcggcat tctcgccgat cagcaatccg atggattgtc cgtggggcga gaaggccttc
     5941 agccgctacc tgggtgaaga ccgtgggcgc tggcgtgagt gggatgccag cgtgctgctg
     6001 gccgaaaccc cggcaggcca atgcccgccg ttgctggtgg atcagggcga ccgtgacgac
     6061 ttcctcgaga agcaactcaa gcctgaagcg ctggaacagg cggcacgcaa aggtggccat
     6121 gcaatgaccc tgcgtatgca gccaggctat gaccatagct actacttcat tgccagcttc
     6181 atcgaggagc acctgcgcca tcatgcg
//
