LOCUS       sequence-174003-        6339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses PchG WT, the thiazolinyl imine reductase from Pseudomonas 
            aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6339)
  AUTHORS   Meneely KM, Ronnebaum TA, Riley AP, Prisinzano TE, Lamb AL
  TITLE     Holo Structure and Steady State Kinetics of the Thiazolinyl Imine 
            Reductases for Siderophore Biosynthesis.
  JOURNAL   Biochemistry. 2016 Sep 27;55(38):5423-33. doi: 
            10.1021/acs.biochem.6b00735. Epub 2016 Sep 15.
  PUBMED    27601130
REFERENCE   2  (bases 1 to 6339)
  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.6b00735"; journalName: "Biochemistry"; date: 
            "2016-09-27- 27"; volume: "55"; issue: "38"; pages: "5423-33"
COMMENT     Sequence Label: pET28b-pchG WT
FEATURES             Location/Qualifiers
     source          1..6339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             669..686
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(739..757)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      753..800
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      837..1292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(924..943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1134..1155
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1384..2199)
                     /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             complement(1384..2199)
                     /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     2107..2126
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2191..2210
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2321..2909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2810..2829
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3063..3080
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3095..3234
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3160..3179)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3320..3342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3336..3527)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4336..5418)
                     /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     5380..5399
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5419..5496)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5702..5721
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5805..5824
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5805..5823
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5824..5848
                     /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             5863..5885
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5904..5921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5931..5948
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 tggcgcgttt catcgagctg ggccgccagt tgcatcgccg gcgcggcatc cgccacctgg
       61 acgcggcctg cggggtgcag gtcggtttcg ccaccctgga catcctcgcc gcgttgctgg
      121 agggcgttgg cccctggtcg ctggagtcgc cctcgaacga cctgtcggcg atgcgcgggc
      181 tgtccctggt gttggcggaa gtgccattga gcctgctcgt gctcaacgaa ctggcggccg
      241 ccgacgacgg gcgcatgacc ttgttgcagc gcgtcagcct gaccaccgat cgcggcacgc
      301 tgagcctgct cagcccccat ggcccgttgt tgtggacgcc tgcggtggcg gtaccggcag
      361 aggatgacga cggcctgttc gcgctgttcg acgagatagc cggcgaaccg ctgcccagcg
      421 cccagctctg gtatgccgaa ccgtgcagct gggcccaggt gcaccagcgc ctgtggccgg
      481 cggcggcggc cgaggcgctg gcgctgctcg ccgacggcga cgaggtgcgc cggcgcaacc
      541 agcgcagcct ggaggtcgcc gccctgtggc agcggatcgg cgagcgcctt ggcttccccg
      601 aggcgccgcc ggcgtcgctg gcgccggcga gcctggaaca ggtgctggag caagcctcgt
      661 gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
      721 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      781 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
      841 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      901 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      961 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1021 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1081 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1141 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1201 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1261 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     1321 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1381 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1441 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1501 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1561 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1621 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1681 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     1741 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     1801 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1861 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     1921 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1981 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2041 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2101 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2161 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     2221 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2281 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2341 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2401 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2461 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2521 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2581 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2641 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2701 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2761 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2821 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2881 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2941 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3001 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3061 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3121 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3181 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3241 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3301 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3361 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3421 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3481 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3541 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3601 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3661 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3721 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3781 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3841 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3901 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3961 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4021 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4081 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4141 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4201 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4261 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4321 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4381 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4441 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4501 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4561 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4621 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4681 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4741 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4801 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4861 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4921 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4981 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5041 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5101 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5161 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5221 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5281 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5341 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5401 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5461 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5521 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5581 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5641 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5701 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5761 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     5821 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     5881 ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc ctggtgccgc
     5941 gcggcagcca tatgagcgac gtccgttccg tggtggtcgc cggttcccgg ttcggccagt
     6001 tctatgccgc cggcgtcgcc gccgatccgc gcttcgtcct gcgcggcatc cttggccagg
     6061 gcagccggcg ttcgcgggcg ttggccgaac gcctcggggt ggagacctgg tgcgaggtcg
     6121 aggcgttgcc cgacgatgtc cgcctggcct gcgtcgcggt cggcggcgcg gcacgcggcg
     6181 agcagggccc ggcgctggcc gaggcgctga tggcgcgtgg catcgacgtg ctgatcgagc
     6241 atccgttgct gccgcgcgaa tggcaggacc tgctgcgcag cgccgagcgc ctgggccggc
     6301 gctgcctgct caacaccttc tatccgcagt tgccggcgg
//
