LOCUS       sequence-341162-        6317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and purification of mature, His-tagged Staphylococcus 
            aureus (strain FPR3757) phosphaatidyl inositol specific 
            phospholipase C (PI-PLC) from E. coli BL21 CodonPlus (DE3) RIL 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6317)
  AUTHORS   Goldstein R, Cheng J, Stec B, Roberts MF
  TITLE     Structure of the S. aureus PI-specific phospholipase C reveals 
            modulation of active site access by a titratable pi-cation latched 
            loop.
  JOURNAL   Biochemistry. 2012 Mar 27;51(12):2579-87. doi: 10.1021/bi300057q. 
            Epub 2012 Mar 16.
  PUBMED    22390775
REFERENCE   2  (bases 1 to 6317)
  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/bi300057q";
            journalName: "Biochemistry"; date: "2012-03-27- 27"; volume: "51"; 
            issue: "12"; pages: "2579-87"
COMMENT     Sequence Label: pSaPIPLC His
FEATURES             Location/Qualifiers
     source          1..6317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1452..1474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1482..1514
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             2442..2459
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2512..2530)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2526..2573
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2610..3065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2697..2716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2907..2928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3091..3195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3196..3264,3265..4056)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3414..3433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4227..4815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4716..4735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4969..4986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5001..5140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5066..5085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5226..5248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5242..5433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6242..6317,1..1007))
                     /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"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggctagc atgactggtg
     1501 gacagcaaat gggtcgcgga tccgaattct cagattcgtt gagtaaaagt ccagaaaatt
     1561 ggatgagtaa acttgatgat ggaaaacatt taactgagat taatataccg ggttcacatg
     1621 atagtggctc attcacttta aaggatccag taaaatcagt ttgggcaaag actcaagata
     1681 aagattacct tacccaaatg aagtcgggag tcaggttttt tgatattaga ggtagagcaa
     1741 gtgctgataa tatgatttca gttcatcacg gcatggttta tttgcatcat gaattaggaa
     1801 aatttctcga tgatgctaaa tattacttga gtgcttatcc aaacgaaaca attgtgatgt
     1861 ctatgaaaaa ggactacgat agcgattcta aagttacgaa gacatttgaa gaaattttta
     1921 gagaatatta ttataataac ccgcaatatc agaatctttt ttacacagga agtaatgcga
     1981 atcctacttt aaaagaaacg aaaggtaaaa ttgtcctatt caatagaatg gggggtacgt
     2041 acataaaaag tggttatggt gctgacacgt caggtattca atgggcagac aatgcgacat
     2101 ttgaaacgaa aattaataat ggtagcttaa atttaaaagt acaagatgag tataaagatt
     2161 actatgataa aaaagttgaa gctgttaaaa atttattggc taaagctaaa acggatagta
     2221 acaaagacaa tgtatatgtg aatttcttga gtgtagcgtc tggaggcagc gcatttaata
     2281 gtacttataa ctatgcatca catataaatc ctgaaattgc aaaaacgatt aaagcaaatg
     2341 ggaaagctag aacgggttgg ctgattgttg actatgcagg atatacgtgg cctggatatg
     2401 atgatatcgt aagtgaaatt atagatagta ataaactcga gcaccaccac caccaccact
     2461 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     2521 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2581 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2641 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2701 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2761 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2821 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2881 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2941 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3001 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3061 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3121 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3181 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3241 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3301 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3361 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3421 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3481 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3541 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3601 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3661 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3721 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3781 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3841 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3901 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3961 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4021 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4081 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4141 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4201 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4261 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4321 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4381 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4441 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4501 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4561 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4621 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4681 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4741 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4801 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4861 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4921 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4981 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5041 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5101 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5161 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5221 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5281 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5341 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5401 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5461 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5521 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5581 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5641 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5701 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5761 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5821 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5881 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5941 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     6001 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     6061 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     6121 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6181 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6241 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6301 acgcgcgggg agaggcg
//
