LOCUS       sequence-173998-        6387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Irp3 WT, the thiazolinyl imine reductase from Yersinia 
            enterocolitica.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6387)
  AUTHORS   Meneely KM, Lamb AL
  TITLE     Two structures of a thiazolinyl imine reductase from Yersinia 
            enterocolitica provide insight into catalysis and binding to the 
            nonribosomal peptide synthetase module of HMWP1.
  JOURNAL   Biochemistry. 2012 Nov 6;51(44):9002-13. doi: 10.1021/bi3011016. 
            Epub 2012 Oct 23.
  PUBMED    23066849
REFERENCE   2  (bases 1 to 6387)
  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/bi3011016";
            journalName: "Biochemistry"; date: "2012-11-6- 6"; volume: "51"; 
            issue: "44"; pages: "9002-13"
COMMENT     Sequence Label: pET28b-irp3 WT
FEATURES             Location/Qualifiers
     source          1..6387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     210..229
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        210..228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    229..253
                     /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             268..290
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             309..326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             336..353
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1461..1478
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1531..1549)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1545..1592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1629..2084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1716..1735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1926..1947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2176..2991)
                     /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(2176..2991)
                     /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     2899..2918
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2983..3002
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3113..3701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3602..3621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3855..3872
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3887..4026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3952..3971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4112..4134
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4128..4319)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5128..6210)
                     /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     6172..6191
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6211..6288)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
       61 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
      121 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
      181 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      241 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
      301 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgc
      361 cgtccgcctc cccaaaacaa cgcgtactga ttgtgggcgc caaatttggc gaaatgtacc
      421 tgaatgcctt tatgcagccc ccggaggggc tggaactggt cggcctgctg gcgcagggaa
      481 gcgcccgctc aagagagctg gcccatgcgt ttggcattcc gctgtatacc tcgccggaac
      541 agataaccgg gatgccggat atcgcctgta ttgtcgtgcg ttcgaccgtc gccggaggag
      601 ctggtacgca gcttgccaga cactttctgg cgcgcggcgt acacgttatt caggagcatc
      661 ccctccaccc ggatgacatc agttccttac aaacgctggc gcaggaacag ggctgctgct
      721 attggataaa cacgttttat ccgcatacgc gcgcaggacg tacatggctt cgcgatgcgc
      781 agcagctgcg tcgctgcctg gctaagacgc cgccggtagt ccacgccacc accagccgac
      841 agttgctcta ctccacgctg gatttgttgt tgctggctct gggcgttgat accgccgccg
      901 tggagtgcga cgtggtcggc agctttagcg attttcactg cctgagactc ttctggccgg
      961 agggagaagc ctgcctgctg cttcagcgct atctcgatcc tgacgatcca gatatgcata
     1021 gccttatcat gcaccgcctg ctgctgggct ggccggaagg tcatctttcg ctggaggcga
     1081 gctacggccc cgtcatctgg tcatccagcc tgtttgtcgc agaccatcaa gagaacgccc
     1141 acagtctcta ccgcagaccg gagatcctgc gcgatccgcc gggtctgacg cgcagcgcgg
     1201 cgcctctgag ctggcgtgac tgctgcgaaa ccgtcgggcc ggagggcgtc agctggttgc
     1261 tacaccagtt gcgcagccat ctggcgggcg aacatccacc agtggcctgc caaaacgtac
     1321 atcagatcgc gttatcacgt ttatggcagc agattttacg taagaccggc aatgcggaga
     1381 tcagacgcct gacgccgccg caccacgacc ggttagccgg tttctacaac gatgatgata
     1441 aggaggcgct gtgactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1501 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1561 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1621 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1681 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1741 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1801 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1861 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1921 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1981 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2041 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2161 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2221 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2281 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2341 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2401 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2461 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2521 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2581 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2641 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2701 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2761 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2821 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2881 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2941 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3001 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     3061 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3121 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3181 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3241 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3301 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3361 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3421 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3481 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3541 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3601 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3661 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3721 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3781 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3841 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3901 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3961 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4021 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4081 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4141 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4201 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4261 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4321 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4381 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4441 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4501 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4561 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4621 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4681 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4741 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4801 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4861 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4921 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4981 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5041 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5101 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5221 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5281 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5341 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5401 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5461 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5521 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5581 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5641 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5701 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5761 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5821 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5881 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5941 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6001 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6061 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6121 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6181 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6241 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6301 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6361 gcaccgccgc cgcaaggaat ggtgcat
//
