LOCUS       sequence-486669-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Thioesterase from Bacillus subtilis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  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 5967)
  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: BsSrfD
FEATURES             Location/Qualifiers
     source          1..5967
                     /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"
     RBS             900..922
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    937..961
                     /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(961..980)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(962..980)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1068..1087)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1293..1370
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1371..2453
                     /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(1390..1409)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3262..3453
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3447..3469)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3555..3697
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3610..3629
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3712..3729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3883..4471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3963..3982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4582..4601)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4593..5408
                     /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             4593..5408
                     /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(4666..4685)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5501..5956)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5638..5659)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5850..5869
                     /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 gagcagcaac tggcctgcgg
      181 tggaacggtt aatgatctcg taaatacgtt cggccacctt ctcggtttcg ctcaggatga
      241 acatgtgacc gtcgctaaat tcatgaaaca ccgggtttcc tgcccacttt ttccaaccat
      301 cagcgtcttt aatgcacttt ttgtccttac gaccattgaa gatatgaacc ggggattgga
      361 tgatgtggct gtcagacgga cggaagcttt ccagtgcacg atagtcagag cggaacgacg
      421 gcagaaagaa agacataact tctttgttct cgacgagctc ttgcggcata ccacctaatt
      481 cgataatgtg cgccaggaac ttttcatcat ccagatgcga gacctttttg cgctccacgt
      541 gaggcggctg aatcgcgctg atgatcacag cctgcgggta aataccttcg cgctcaagtt
      601 tctgcgccag gcgaaacgca accatgccgc ccatgctgtg accaaacaaa acaaatggac
      661 gatccggatg cagattcaat tcttgtttat acaggctaac caattgctcg aaatcctcca
      721 ctggggacat ctggttcgtg ccgtgacccg gcggttcagc ggccagcatc tcgcactcac
      781 cttgcagata ggtgtgcaac ggtctgaagc tcgcgctgta gccgcccgcg aacggaaaac
      841 aaatcagctg ggtcttctca gttgtatcaa atgatttgaa tagttgtacc atatgtatat
      901 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
      961 ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg
     1021 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     1081 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     1141 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     1201 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     1261 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     1321 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     1381 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     1441 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     1501 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     1561 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     1621 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     1681 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     1741 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     1801 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     1861 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     1921 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     1981 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     2041 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     2101 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     2161 ttggtgcgga catctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     2221 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     2281 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     2341 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2401 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2461 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     2521 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2581 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2641 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2701 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2761 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     2821 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2881 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     2941 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     3001 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     3061 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3121 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3181 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3241 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3301 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3361 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     3421 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     3481 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     3541 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     3601 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat
     3661 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     3721 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3781 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3841 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3901 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3961 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4021 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4081 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4141 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4201 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4261 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4321 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4381 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4441 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4501 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt
     4561 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt
     4621 gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     4681 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     4741 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     4801 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     4861 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat
     4921 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     4981 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     5041 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     5101 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     5161 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     5221 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     5281 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     5341 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     5401 ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5461 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt
     5521 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5581 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5641 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5701 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5761 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5821 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5881 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5941 ccgctacagg gcgcgtccca ttcgcca
//
