LOCUS       sequence-372578-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses salicylate adenylation enzyme PchD from Pseudomonas 
            aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  AUTHORS   Shelton CL, Meneely KM, Ronnebaum TA, Chilton AS, Riley AP, 
            Prisinzano TE, Lamb AL
  TITLE     Rational inhibitor design for Pseudomonas aeruginosa salicylate 
            adenylation enzyme PchD.
  JOURNAL   J Biol Inorg Chem. 2022 May 5. pii: 10.1007/s00775-022-01941-8. doi:
            10.1007/s00775-022-01941-8.
  PUBMED    35513576
REFERENCE   2  (bases 1 to 6973)
  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: "J Biol 
            Inorg Chem. 2022 May 5. pii: 10.1007/s00775-022-01941-8. doi: 
            10.1007/s00775-022-01941-8."
COMMENT     Sequence Label: pET28b-pchD WT
FEATURES             Location/Qualifiers
     source          1..6973
                     /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(1851..1868)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1878..1895)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1914..1936
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1951..1975
                     /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(1975..1994)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1976..1994)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2078..2097)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2303..2380
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2381..3463
                     /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(2400..2419)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4272..4463
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4457..4479)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4565..4704
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4620..4639
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4719..4736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4890..5478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4970..4989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5589..5608)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5600..6415
                     /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             5600..6415
                     /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(5673..5692)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6507..6962)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6644..6665)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6856..6875
                     /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 gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcctcatgcg cgggcctcca gctcgcggcg caggcgctcg
      241 cgcaggtcct tcttgctgat cttgccgatg ccggtttggg ggaaggccgg gaccagctcg
      301 atgcggtccg gcaccttgaa cgcggccagc ccgcaggcgt gcaggtattg cttcagcttc
      361 agcgccgagg gcgccggctg gcgcgggatg acgaaggcgc aggtgcgctc gccgagcagg
      421 ctgtcgggca tcgccacgac ggtggcgtcg tgcacctgcg gatgggctat cagcaggttc
      481 tccacctctt ccgcggcgat cttctcgccg ccgcggttga tctggtcctt gtcgcggccc
      541 tccaccacca ggtagccgtc cttgtcgcgg ctgacccggt cgccggtgcg gtagaagccg
      601 tccgcgctga aggccttggc gttgtgttca ggcaggcggt agtagccgcg gatggtgtag
      661 ggaccgcgca cggtcagttc gcccacctcg ccggggccga cctcgcggcc ctcggcgtcg
      721 accacccgca cttcgtcgtc cggcgacagc ggccggccct gggtatgcag cacgcgttcg
      781 ggcggatcgt ccaggcgggt gtagcagatc agcccctcgg ccattcccag cacctgttgc
      841 aactggcagc cgagcaccgg ttcgacgcgt tgcgcggcac tgctgccgag cctggagccg
      901 ccgacctgga gcagacgcag gctggataga tcggcgcggc ggctttcctg ggcatccagc
      961 cagagcatcg ccagcggcgg taccagggcg gtgtgggtga cccgttcacg ggcgatcagg
     1021 gcgaagcaat gctcgggatc ggcgcgctgg ctgaccacca cgcggccgct ggccagcagc
     1081 gtgccgatca ctccggggca gcagagagtg aagttgtgcg ccatcggcaa gccggtgagg
     1141 tacaccgtgt gctcgtcgaa gccgcagacc tcggcgctgg cgcggacgtt gtagagatac
     1201 tcgcgatgac gccggggaat cagcttcggc gtgccggtgg tgccgccgga caactggaag
     1261 caggcgatat cctcggcgcg ggcggcgcag tcttccagcg cgtcggcctg gtacagcggt
     1321 gccagcgctt gcagcggcgc ctcggcttcg ccgtggatca gcgccatccg gcaggcgccg
     1381 ctggcgagca gctcacgggc catcggccgt gggtcgaagc cgtcgatccg ttcggcgccg
     1441 atgtaggcct tggcctcggc gaagcgacag aaaccgctga tctcatgctg gcgatgggcg
     1501 gggagggcga gcaccgggcg tacgccgagc tggaacaggg cgaagcaggt ctcgacgaag
     1561 gcgatgccgt tgggcaggtg cagcactacc gtgtcgccat gtgcgaggcc ggcctggcgc
     1621 agtccggctg ccaggcggcg gcagcgctgg agcaggtcgg catagctgag ccgctggtcg
     1681 tcgtcgcaga gcgcgatccg ccgcggatgg cgcgcggcgc tggtggccag ggcttcggcg
     1741 aagctctggt cctgccagtg gccggcgtcg agatagcggc ggacgaaggc ggcgggccag
     1801 tcgggggcgt cgtcgacggc cgatggggtg acgggcgagg aagtcatatg gctgccgcgc
     1861 ggcaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt
     1921 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
     1981 agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg
     2041 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     2101 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     2161 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     2221 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     2281 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     2341 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     2401 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     2461 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     2521 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     2581 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     2641 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     2701 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     2761 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     2821 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     2881 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     2941 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     3001 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     3061 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     3121 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     3181 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     3241 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     3301 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     3361 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     3421 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     3481 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     3541 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     3601 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     3661 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     3721 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     3781 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     3841 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     3901 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3961 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     4021 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     4081 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     4141 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     4201 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     4261 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     4321 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     4381 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     4441 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4501 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4561 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     4621 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     4681 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5521 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     5581 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     5641 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5701 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     5761 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5821 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     5881 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     5941 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     6001 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     6061 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     6121 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     6181 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     6241 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     6301 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     6361 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     6421 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6481 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     6541 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     6601 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     6661 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     6721 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     6781 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     6841 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     6901 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     6961 gtcccattcg cca
//
