LOCUS       sequence-174008-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses PchA WT, the isochorismate synthase from Pseudomonas 
            aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  AUTHORS   Meneely KM, Luo Q, Dhar P, Lamb AL
  TITLE     Lysine221 is the general base residue of the isochorismate synthase 
            from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion 
            limited.
  JOURNAL   Arch Biochem Biophys. 2013 Oct 1;538(1):49-56. doi: 
            10.1016/j.abb.2013.07.026. Epub 2013 Aug 11.
  PUBMED    23942051
REFERENCE   2  (bases 1 to 6715)
  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: 
            "10.1016/j.abb.2013.07.026"; journalName: "Arch Biochem Biophys"; 
            date: "2013-10-1- 1"; volume: "538"; issue: "1"; pages: "49-56"
COMMENT     Sequence Label: pET28b-pchA WT
FEATURES             Location/Qualifiers
     source          1..6715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             408..425
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(478..496)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      492..539
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      576..1031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(663..682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     873..894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1123..1938)
                     /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(1123..1938)
                     /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     1846..1865
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1930..1949
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2060..2648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2549..2568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2802..2819
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2834..2973
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2899..2918)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3059..3081
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3075..3266)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5099..5118
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5138..5215)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5421..5440
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5524..5543
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5524..5542
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5543..5567
                     /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             5582..5604
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5623..5640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5650..5667
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 tgaacacgcc gatccgcgcc cgcctcgctg acgcaggcgg catcctgcgg ctgctacaag
       61 cgctgcatcc gacccccgcg gtgggcggct acccacgcag cgcggcgctg gactacatcc
      121 gccagcacga agggatggac cgcggctggt acgccgcgcc gctgggctgg ctcgacggcg
      181 aaggcaacgg cgatttcctg gtggcgctgc gctcggccct gctcacgccg ggccggggct
      241 acctgttcgc cggctgcggt ctggtaggcg attcggaacc ggcccacgag tatcgcgaaa
      301 cctgccttaa gctcagtgcc atgcgggaag ctctatccgc cataggcggc ctggacgaag
      361 tgcccttgca gcgcggcgtc gcctgaaagc ttgcggccgc actcgagcac caccaccacc
      421 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      481 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      541 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      601 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      661 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      721 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      781 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      841 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      901 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      961 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1021 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1081 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     1141 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1201 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1261 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1321 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1381 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     1441 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     1501 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     1561 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     1621 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     1681 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     1741 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     1801 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     1861 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     1921 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     1981 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2041 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2101 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2161 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2221 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2281 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2341 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2401 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2461 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2521 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2581 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2641 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2701 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2761 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2821 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2881 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2941 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3001 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3061 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3121 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3181 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3241 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3301 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3361 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3421 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3481 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3541 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3601 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3661 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3721 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     3781 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     3841 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     3901 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     3961 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     4021 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4081 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4141 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4201 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     4261 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     4321 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     4381 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     4441 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     4501 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4561 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     4621 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4681 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4741 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4801 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4861 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4921 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4981 tttgcccgcc agttgttgtg ccacgctccg ccatcgccgc ttccactttt tcccgcgttt
     5041 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5101 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5161 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5221 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5281 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5341 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5401 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5461 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     5521 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5581 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     5641 agcagcggcc tggtgccgcg cggcagccat atgagccggc tggcgcccct gagccagtgc
     5701 ctgcacgcct tgcgcggcac cttcgagcgc gccatcggcc aggcgcaggc gctcgatcgt
     5761 ccggtgctgg tggcggcatc gttcgagatc gacccattgg acccgctgca ggtattcggt
     5821 gcctgggacg accggcaaac gccctgcctg tactgggaac agcccgagct ggcgttcttc
     5881 gcctggggct gcgccctgga gctgcaaggc cacggcgaac agcgcttcgc ccggatcgag
     5941 gaaaactggc aattgctctg cgccgacgcc gtggtcgagg gcccgctggc gccgcgcctg
     6001 tgcggcggat tccgcttcga tccgcgcggc ccgcgcgagg aacactggca agccttcgcc
     6061 gatgccagcc tgatgctcgc cggcatcacc gtgctgcgcg agggcgaacg ctaccgggta
     6121 ctctgccaac acctggccaa gcccggcgaa gatgccctgg ccctggccgc ctaccactgc
     6181 tcggcgctac tgcgcctgag gcagccggcc agacgccggc cctcggggcc gaccgctggc
     6241 gcgcagggcg acgcttcggc gcaggagcgc aggcaatggg aagccaaggt gagcgacgcg
     6301 gtaagcagtg tccgccaggg acgcttcggc aaggtcgtgc tggcccgcac ccaggcccgg
     6361 cctctcggcg acatcgagcc gtggcaggtc atcgaacacc tgcgtctgca acatgccgac
     6421 gcccagctgt tcgcctgtcg ccgcggcaac gcctgcttcc tcggcgcctc cccggaacgc
     6481 ctggtccgca ttcgcgccgg cgaggcactc acccatgccc tggccgggac catcgcccgc
     6541 ggcggcgatg cccaggaaga tgcgcggctc ggacaggccc tgctggacag cgccaaggac
     6601 aggcacgaac accagttggt ggtggaggcg atccgtacgg ccctggaacc cttcagcgag
     6661 gtgctggaaa tccccgatgc gcccggcctg aaacgactgg cgcgagtcca gcacc
//
