LOCUS       sequence-486670-        7719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adenylase-epimerase didomain of the nonribosomal peptide synthetase 
            PchE from Pseudomonas aeruginosa for the production of pyochelin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7719)
  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 7719)
  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: PaPchE-AE
FEATURES             Location/Qualifiers
     source          1..7719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2597..2614)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2624..2641)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2660..2682
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2697..2721
                     /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(2721..2740)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2722..2740)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2824..2843)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3049..3126
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3127..4209
                     /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(3146..3165)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5018..5209
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5203..5225)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5311..5450
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5366..5385
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5465..5482)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5636..6224)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5716..5735)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6335..6354)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6346..7161
                     /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             6346..7161
                     /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(6419..6438)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7253..7708)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7390..7411)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7602..7621
                     /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 gagtcaaagt gcgcgggtca
      181 tgctctcggc caggcggcga cgatcgacct tgccattgcc gttcaacggc aggcttggca
      241 ggcaccacag gcgttcgggc cgcatcgcct ggggcaggcg ttgctccagg ccggcctgga
      301 gcgcggcggc gtcgagcccg agtcccggtg ccagcgcctc gaccagggcg atccgctcgc
      361 tgcgccacag gcaacgcggc gccagtcccg cagcggcgag atcggccaac aggctcggca
      421 gctccgccag gcgcagcggc cggtcgtcga gcaaggccgc accgagcagc gccagtggcg
      481 actcgcatag caggtccacc agcaacaggc ggccctgcgg gcgcagcagc gccgccgcca
      541 gcgccaggcc ttcgcggctg gcctcgtagg cgtgcagcgc ggcgaagctg atcacccggt
      601 cgtagcgacc gaggtgctcg gcaggtagca ggccgtcgtc cagcgcctgc accacgatcc
      661 gttccggcaa gcgggtggcg gcggcgtcga ggaggacgcg gctgcgttcg aagagggtca
      721 gttccagccc tgggcgcaac agcgaggcca tgccctggtc gagccagagc ccggcgcggg
      781 tatccagcac cgccacccgc aggcgttcgc cggcagccgg cgtcggcagc gcttcgagca
      841 ggcgcgccag ggcctcgcgg ccgtccggca ggcgcgcggc caccgcctgc ggcgccagcc
      901 agggatcgag gagcagcgcg gccggcgcct gttcgccggc gagcaggccg cgcagagcgt
      961 tgcccatcgc ggtcagcccc gcggcctgcc agccgagccg gcgcaacgcc tcgtcgagcg
     1021 acggcagcgc gctggcggcg gagtccgcta gccagtccag ccagcgccgg cgcaaaccgt
     1081 cgtctagctc cagcggcgcc tccagcagcg cgccgaggat ttcccgggtg agtaccgcct
     1141 cggcctctgc caggccggcg aagggctgtg cggccggtag atccatggag ccttcggccc
     1201 gtggcgccag gcgcggtacc agcaccgcgc cgaggctcgc cacgccaccg ccgagcaccg
     1261 ccgcgcaggc gctctccacg ccagcctggg cgcacagcgc ggcctccacc tcgcccaact
     1321 cgatgcgctg gccgcgcact ttcacctgct ggtcgacccg accgaggaat tccagggtac
     1381 cgtcgcccca gtagcgaccg cgatcgccgg tgcgatacca gcggccctgg gcatcgtgga
     1441 cgaaacgccg ggcgctgagt tcgggatcgt tgcgatagcc gcgggccagg ctggcgccgc
     1501 cgatccacag ctcgccgacc accaggtccg gcacgtcgcg cccgtgggtg tcgaccaccc
     1561 ggtaggcctg tcccggcaat ggccggccgt agggaatcga acgccagtgc ggcggcaccg
     1621 tatcgacgct ctgcaggttc gaccagatgc ccgcttcggt agcgccaccc agcacatgca
     1681 ggcggcagcc ttcggcacaa cgtgggcgca ggcggccggg caggtccagg gccacccagt
     1741 cgccggacag cagcaccgcc cgcagactgc gatagtcggc ctggctcgcc ggcaggctga
     1801 gggccatctc cagcaaggcc ggcgccgagt tccacaggct caccgcatgc cgctggatag
     1861 cctccgccca ggcagcggca tcgcgcgcct gttcctgggc cggcaggacc aggctggcac
     1921 cggcgccgag gccgccgaac aggtcgaaga ccgacagatc gaagtccagc gcggagaccg
     1981 ccagcaagcg atccgatgcg ttcacccgca gcaggtcgag cagcgcgtcg atggtattga
     2041 tcgccgccgc gtggctgacc tcgacgccct tgggcacccc ggtggagccc gaggtgtaga
     2101 tcacataggc actcgcctgc ggcgccagcg gcacgggggc ggccagcgcc gggccgcgca
     2161 gcaggcgctg gacatccagg cgcggcggca aggcctgcgg atcgtcctcc tcggtgatcg
     2221 ccaggcatac cccggcggcc tcttcgatca ggcgccgccg tgcgggcggc tggtcgatgt
     2281 ccagcggcac gtagcaggcg cctgcggcga gcacgccgaa taccgccgcg acctgctgcg
     2341 gtccgcgcgg caggctgacc tcgaccgcgt cgccagggcg caccccggct tcgcgcaggc
     2401 cgccggcgat gcgcagcgca cgctcggcca gttcgccgcg ggtgacacgt tggtcgcgat
     2461 agagcagcgc gtcggcatcc ggcgcctcgg cggcgcgaag gaagaagtcg cgatgcaggg
     2521 tgcgcgccgt ggcgcatgcc ggctggccgt tgagcagggc ccggcgcgcc tgctgcgccc
     2581 agggcaacgg catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat
     2641 ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
     2701 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct
     2761 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
     2821 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     2881 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     2941 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     3001 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
     3061 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     3121 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     3181 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     3241 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     3301 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     3361 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     3421 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     3481 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     3541 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     3601 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     3661 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     3721 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     3781 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     3841 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     3901 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     3961 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     4021 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     4081 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     4141 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4201 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     4261 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     4321 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     4381 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     4441 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     4501 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
     4561 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     4621 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     4681 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     4741 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     4801 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     4861 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     4921 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     4981 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     5041 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     5101 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     5161 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     5221 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     5281 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     5341 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5401 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     5461 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5521 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5581 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5641 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5701 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5761 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5821 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5881 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5941 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6001 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6061 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6121 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6181 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6241 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6301 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     6361 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
     6421 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     6481 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     6541 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     6601 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     6661 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     6721 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     6781 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     6841 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     6901 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     6961 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     7021 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     7081 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     7141 atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta
     7201 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     7261 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     7321 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     7381 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     7441 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     7501 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     7561 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     7621 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     7681 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
