LOCUS       sequence-341161-        4483 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression and purification of the mature form of Bacillus 
            thuringiensis phosphaatidyl inositol specific phospholipase C 
            (PI-PLC), UniProt ID P08954 from E. coli BL21 CodonPlus (DE3) RIL 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4483)
  AUTHORS   He T, Gershenson A, Eyles SJ, Lee YJ, Liu WR, Wang J, Gao J, Roberts
            MF
  TITLE     Fluorinated Aromatic Amino Acids Distinguish Cation-pi Interactions 
            from Membrane Insertion.
  JOURNAL   J Biol Chem. 2015 Jul 31;290(31):19334-42. doi: 
            10.1074/jbc.M115.668343. Epub 2015 Jun 19.
  PUBMED    26092728
REFERENCE   2  (bases 1 to 4483)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M115.668343"; journalName: "J Biol Chem"; date: 
            "2015-07-31- 31"; volume: "290"; issue: "31"; pages: "19334-42"
COMMENT     Sequence Label: pBtPIPLC_N168C His
FEATURES             Location/Qualifiers
     source          1..4483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(38..829,830..898))
                     /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     661..680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(899..1003)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1029..1484)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1166..1187)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1378..1397
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1521..1568)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1564..1582
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1635..1652)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2563..2585
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2616..2635)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2617..2635)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3144..3335
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3329..3351)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3437..3576
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3492..3511
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3591..3608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3762..4350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3842..3861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
       61 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
      121 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
      181 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
      241 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
      301 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
      361 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
      421 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
      481 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
      541 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
      601 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
      661 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
      721 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
      781 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
      841 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
      901 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
      961 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     1021 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1081 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1141 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1201 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     1261 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     1321 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     1381 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     1441 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg
     1501 gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt
     1561 tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta
     1621 gcagccggat ctcagtggtg gtggtggtgg tgctcgagtt cttttattaa tgacttattc
     1681 gctcttataa cttcttgata caataatggt gaccactttt catttatgta gtcttgaatt
     1741 acccagccta ctcttgtagg attcttttgt tttatatcgt ttgcaatttc aggatttata
     1801 taagaagcgt agtaatatgg actattccat gctgtaccac cagaagacaa gcttgtaaaa
     1861 ttaatatata gatgatttaa atcctcgcta ttgttcatcg tttcatccat cgtatcttta
     1921 atagatttta ctttctcatc ataattcact ttatatttat cttgtactgt tacatttaca
     1981 ttttggttta cagttgtggt aaacgtctca ttatctggcc aataaaaatt attatatcct
     2041 ccagattcac aactaccact atatcttttt agtagtacaa ttttcccacg agcatctcca
     2101 agttttatat ttccttctgt ttttaaaaag ataggatcaa caaaataatt tttttcaaac
     2161 gtactactaa atgaaccttc tgcccctttc atatcctcat actctttttt taaagacata
     2221 ataattgttt cactcgggtt atcttttaaa aattgtttcg cttcatttat gaattcatgc
     2281 agtgttacgt aaagatataa tggcccatga tgaagaacta tcgtattatc atctgttaaa
     2341 cgtcctctta tatcaaaaat gcgagctcca tggtccattt gatagcgaaa atcatattct
     2401 tgcgtcattc cccacacttg cttaatcgga ttttgcaact tgaacgtccc actatcgtgt
     2461 gttcctggaa ttgaaattcg tgctaacggg atattatcag gtataggttg catccatttt
     2521 gaccaatttt caagctcatt aacagagcta gccatatgta tatctccttc ttaaagttaa
     2581 acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg tattaatttc
     2641 gcgggatcga gatctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc
     2701 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2761 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2821 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2881 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2941 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3001 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3061 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3121 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3181 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3241 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3301 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3361 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3421 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3481 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     3541 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3601 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3661 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3721 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3781 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3841 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3901 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3961 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4021 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4081 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4141 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4201 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4261 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4321 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4381 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4441 atcttcacct agatcctttt aaattaaaaa tgaagtttta aat
//
