LOCUS       sequence-486686-        8760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nonribosomal peptide synthetase from Pseudomonas protegens for the 
            production of enantiopyochelin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8760)
  AUTHORS   Ronnebaum TA, Meneely KM, Horsman GP, Prisinzano TE, Lamb AL
  TITLE     Stuffed Epimerase Domains of Pyochelin Biosynthesis are Defunct 
            Methyltransferases.
  JOURNAL   Biochemistry. 2025 Sep 16;64(18):3919-3934. doi: 
            10.1021/acs.biochem.5c00172. Epub 2025 Aug 26.
  PUBMED    40857142
REFERENCE   2  (bases 1 to 8760)
  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"; doi: 
            "10.1021/acs.biochem.5c00172"; journalName: "Biochemistry"; date: 
            "2025-09-16- 16"; volume: "64"; issue: "18"; pages: "3919-3934"
COMMENT     Sequence Label: PpPchE
FEATURES             Location/Qualifiers
     source          1..8760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3638..3655)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(3665..3682)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3701..3723
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3738..3762
                     /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(3762..3781)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3763..3781)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3865..3884)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4090..4167
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4168..5250
                     /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(4187..4206)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6059..6250
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6244..6266)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6352..6491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6407..6426
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6506..6523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6677..7265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6757..6776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7376..7395)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7387..8202
                     /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             7387..8202
                     /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(7460..7479)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(8294..8749)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8431..8452)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8643..8662
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttacagc acgccttcaa
      181 ccatcgcgtt ctggttcacc ggacgttgct cacgacggct cgccagcagt tgaccctgca
      241 gcgcaacggt cgccgcgtta aagaagtctt tcaggctcag ttccagttga aactgacgcg
      301 caatacgctc aatcagacgc gccgccagca ggctgttacc gcccagcgca aagaagccct
      361 ggtgacgacc cacttgcgga acttccagca gctcctgcca cagcgccgcc accgcttcct
      421 cccattcacc ttgcggcggc tccgcgcttt gcggttgaat accacgacgt tgctcctgca
      481 gcagccacag cagcgccgca cgatcaacct tgccgttggt gctcagcggc agttccggca
      541 ggctcagcca cagatccggc agcatgtgct ccggcagcca ctggcccagc cattgacgca
      601 gcgcctgcgg ttccggcagc ggctcacgcg ccagcagcac cgccgccagc tgcgcctcgc
      661 tacccggcag aaccaggctc agcgcacgat caatcgccgg gtgacgctcc agcgccacgt
      721 caatttccgc cagctcgata cgaaagccac gcactttaac ctggtgatcc agacgaccca
      781 ggaattccag gctcgcatcc gcccaataac ggccctggtc accggtacgg taccaacgac
      841 gaccttcgtg ctcaacgaaa cgttgcgcgc tcagggtcgg caggccacga taacccgccg
      901 ccacgcccgc accgccgatc cacagctcgc ccggaaccca atccgggcag tcacgaccct
      961 ggctatcaac cacacgaaag cattggttgt ccagcggacg gccatacgga atgctacgcc
     1021 agtgacccgg cacttgcgca acgtcctggt agttgctcca gatcgccgct tcggtcgcac
     1081 cgcccagcgc cacaaacgga acacgcagac ccgccgcacg ctccagacga cgcggcagat
     1141 ccagaccaac ccagtcaccg ctaaccatcg ccagacgcag cgcacccaga ccgccatcat
     1201 cacgaacctg cagggtcagc atatccagca gcgccggaac gctgttccac acgctaacac
     1261 gttgttcacg aatcagacgc agccattcac gcggctcttt acgcagacca tccgccggca
     1321 gcaccagcgc accgccaacg ctcagcagac cgaacaggtc atacacgctc agatcgaagt
     1381 ccagcgcgct cacacccaga acacggtctt ccgcgctaac ttggtaacga cggttaatcg
     1441 cggtgatggt gttcatcgcc gcttggtggc tcagttccac gcccttcggt tcaccggtgg
     1501 taccgctggt gaagataaca tacgccagcg cgtccgccgc catctccagc ggctgaccca
     1561 gcgccggcgc aaccagcgct tgcgccaggg taaccacacg cagttccgcc agccaaatac
     1621 cgccctcacg gtgcgcctga tcggtgataa ccacacgcgc gcccgcacgc tgataaatca
     1681 tgtcacgacg cgccggcggt tgctcaatac ccaccggcac gtacaccgca cccgccgcca
     1741 gggtacccag caccgcaacc acctgatcac gacccttcgg cagggtaatc gccaccgcat
     1801 cacccggttg aacgccccat tcacccagcg cacccgccag ctgcagcgcc tgacgcgcca
     1861 gctccgcata gctcaggcta ccttgttcgc agatcagcgc aaccgcctcc gggttcagac
     1921 gcgcttgttg aaagaaacct tggtgcagca gctgcgccgg ggtcgcacgc gcggtcgcgt
     1981 tcacctgttg acgacgctgt tgctgcgccg ccggcagcgg cagcggcgcc acttgctgcc
     2041 agtcgttcgc gcacagccac agcagcagcg cacgatacgc atcaaacatt tcgtccagca
     2101 ggccctccgg gaacagcgca tccaccgcat cccagttcag cagcagacca tccgccagcg
     2161 ggtagctctg gtggtccagc caaacttgcg gggtctggct cagcgcccaa cccagttcgc
     2221 ccagctgctc acggcaggtc gcatcaacaa acgccggacc caggttgcac gcgaaaacca
     2281 ccggcgcacc ttgaccgcca ccctgacgca gacgcgcacg ttcacgcagc acctcaacac
     2341 ccggccacgc gctatgcgca atatcacgat gcagttgcgc ctgcagcgcc agcgcctgtt
     2401 ggctaaagtg cgccggttga cgctgatcca cttccagcag aaccaggttg ctgaagtccg
     2461 ccagcaggtg cggcacgcac gggtgcagtt cctgacggtt aaacagcgga acgttcagca
     2521 ggaagtgctg gctcgcgctc caacgaccca gcacttgcgc atacgcgcac gccagaacgc
     2581 tcgccagggt cagaccatgt tgacgcgcac gttgttccag cgcttgcagc tgaccctggt
     2641 ccagacgcat cgccagacga cgaaaacgcg cggtacccgc gttttgcggt tcctgcgcca
     2701 gcggcagctg cggaccatcc ggcagttcct ccagacgctc cagccaatag tcacgcgccg
     2761 ccgccagcag cggcgcacgc gccgcttgtt ccgcacgcag gtaatccgca aagttccagt
     2821 ccagcgccgg caggctgtgc tcttgacccg catgaatcag cgccaggtca cgcagcagaa
     2881 taccgatgct cagcacatcc gcaaccagca ggtcgatgtt caggtgcaga cggctacggc
     2941 cctccggcag caggctcagt tgcacatcaa aaacttgacc acggctaact tccaggcaac
     3001 ggtggctcag gttctcacga atctgttcca gcgcctccgc cgcaccaacg cagctcgctt
     3061 gacgcaggtc atgaacggtc agacccggcc acgggctctg cggcagaata cgttggcagg
     3121 tatcgctgat gaagcacgca cgcagcatcg ggtgacgctg gcacagacga cgcagcgctt
     3181 gttccagacg ctgcggctcc agaccatgac catccagctc ctgatacagg tggcaaccca
     3241 cgccacccag cggcataccc ggatcacgac caaaccagta cgcttgctgc agcggggtca
     3301 gcgcaaagtc gctgttcgcc ggcgcgctcg caaccgcttg cagacgcggc gccgccaggc
     3361 cacgacgcgc ctcaatcagc gcccaccacg catccagggt cggttgctgg atcagttccg
     3421 cgaagctcag cgccacaccc tccgccgcca gcagcgccgg cagacgcata atgctaacgc
     3481 tatccaggcc ctgttcgatc aggctcgctt gcggttccag gtcctccgcg ctgtaaccca
     3541 gcagacgcgc aataccctcg cacagaccct gcgggctgtg gctcagaccc gcatccgccg
     3601 ggtgcgcacg cgccagcgcc gggttgtcgg tcatatggct gccgcgcggc accaggccgc
     3661 tgctgtgatg atgatgatga tggctgctgc ccatggtata tctccttctt aaagttaaac
     3721 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
     3781 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     3841 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     3901 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     3961 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     4021 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     4081 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     4141 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4201 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4261 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4321 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4381 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4441 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4501 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4561 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4621 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4681 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4741 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4801 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4861 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4921 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4981 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     5041 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     5101 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     5161 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5221 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     5281 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     5341 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     5401 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     5461 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     5521 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     5581 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     5641 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     5701 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     5761 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     5821 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     5881 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     5941 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     6001 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     6061 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     6121 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     6181 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     6241 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     6301 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     6361 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     6421 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     6481 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6541 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6601 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6661 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6721 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6781 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6841 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6901 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6961 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7021 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7081 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     7141 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7201 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7261 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7321 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     7381 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     7441 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     7501 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     7561 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     7621 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     7681 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     7741 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     7801 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     7861 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     7921 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     7981 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     8041 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     8101 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     8161 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     8221 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8281 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     8341 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     8401 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     8461 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     8521 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     8581 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     8641 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     8701 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
