LOCUS       sequence-345681-        4483 bp    DNA     circular SYN 15-DEC-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 Dec 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 His
FEATURES             Location/Qualifiers
     source          1..4483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2392..2531
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2457..2476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2617..2639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2633..2824)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3333..3352
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3333..3351
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3383..3405
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4316..4333
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4386..4404)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4400..4447
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     2461 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     2521 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2581 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2641 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     2701 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     2761 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     2821 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     2881 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     2941 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     3001 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     3061 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     3121 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     3181 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     3241 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     3301 caacgctgcc cgagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca
     3361 acggtttccc tctagaaata attttgttta actttaagaa ggagatatac atatggctag
     3421 ctctgttaat gagcttgaaa attggtcaaa atggatgcaa cctatacctg ataatatccc
     3481 gttagcacga atttcaattc caggaacaca cgatagtggg acgttcaagt tgcaaaatcc
     3541 gattaagcaa gtgtggggaa tgacgcaaga atatgatttt cgctatcaaa tggaccatgg
     3601 agctcgcatt tttgatataa gaggacgttt aacagatgat aatacgatag ttcttcatca
     3661 tgggccatta tatctttacg taacactgca tgaattcata aatgaagcga aacaattttt
     3721 aaaagataac ccgagtgaaa caattattat gtctttaaaa aaagagtatg aggatatgaa
     3781 aggggcagaa ggttcattta gtagtacgtt tgaaaaaaat tattttgttg atcctatctt
     3841 tttaaaaaca gaaggaaata taaaacttgg agatgctcgt gggaaaattg tactactaaa
     3901 aagatatagt ggtagtaatg aatctggagg atataataat ttttattggc cagataatga
     3961 gacgtttacc acaactgtaa accaaaatgt aaatgtaaca gtacaagata aatataaagt
     4021 gaattatgat gagaaagtaa aatctattaa agatacgatg gatgaaacga tgaacaatag
     4081 cgaggattta aatcatctat atattaattt tacaagcttg tcttctggtg gtacagcatg
     4141 gaatagtcca tattactacg cttcttatat aaatcctgaa attgcaaacg atataaaaca
     4201 aaagaatcct acaagagtag gctgggtaat tcaagactac ataaatgaaa agtggtcacc
     4261 attattgtat caagaagtta taagagcgaa taagtcatta ataaaagaac tcgagcacca
     4321 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     4381 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     4441 ttttttgctg aaaggaggaa ctatatccgg attggcgaat ggg
//
