LOCUS       sequence-237447-        8069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of PIS, PI4K and PI3P5K mutant E545K.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8069)
  AUTHORS   Botero S, Chiaroni-Clarke R, Simon SM
  TITLE     Escherichia coli as a platform for the study of phosphoinositide 
            biology.
  JOURNAL   Sci Adv. 2019 Mar 27;5(3):eaat4872. doi: 10.1126/sciadv.aat4872. 
            eCollection 2019 Mar.
  PUBMED    30944849
REFERENCE   2  (bases 1 to 8069)
  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"; journalName: "Sci Adv."; 
            date: "2019-03-27"; volume: "5(3):eaat4872. doi"; pages: " 
            10.1126/sciadv.aat4872. eCollection 2019 Mar"
COMMENT     Sequence Label: p15aC-3D1D-5
FEATURES             Location/Qualifiers
     source          1..8069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..660)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     575..594
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(661..765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     833..851
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     RBS             1025..1033
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(4391..4408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4525..5070)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4605..4624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             5346..5354
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5362..5391
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     RBS             6082..6090
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6102..6131
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
ORIGIN
        1 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
       61 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
      121 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
      181 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
      241 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
      301 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
      361 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
      421 accgtctttc attgccatac ggaactccgg atgagcattc atcaggcggg caagaatgtg
      481 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
      541 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
      601 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
      661 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
      721 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      781 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      841 tatcacgagg ccctttcgtc acacagctaa caccacgtcg tccctatctg ctgccctagg
      901 tctatgagtg gttgctggat aactttacgg gcatgcataa ggctcgtata atatattcag
      961 ggagaccaca acggtttccc tctacaaata attttgttta actttacaac acgaacgaaa
     1021 aacataagga ggtacataat gcctccacga ccatcatcag gtgaactgtg gggcatccac
     1081 ttgatgcccc caagaatcct agtagaatgt ttactaccaa atggaatgat agtgacttta
     1141 gaatgcctcc gtgaggctac attaataacc ataaagcatg aactatttaa agaagcaaga
     1201 aaataccccc tccatcaact tcttcaagat gaatcttctt acattttcgt aagtgttact
     1261 caagaagcag aaagggaaga attttttgat gaaacaagac gactttgtga ccttcggctt
     1321 tttcaaccct ttttaaaagt aattgaacca gtaggcaacc gtgaagaaaa gatcctcaat
     1381 cgagaaattg gttttgctat cggcatgcca gtgtgtgaat ttgatatggt taaagatcca
     1441 gaagtacagg acttccgaag aaatattctg aacgtttgta aagaagctgt ggatcttagg
     1501 gacctcaatt cacctcatag tagagcaatg tatgtctatc ctccaaatgt agaatcttca
     1561 ccagaattgc caaagcacat atataataaa ttagataaag ggcaaataat agtggtgatc
     1621 tgggtaatag tttctccaaa taatgacaag cagaagtata ctctgaaaat caaccatgac
     1681 tgtgtaccag aacaagtaat tgctgaagca atcaggaaaa aaactcgaag tatgttgcta
     1741 tcctctgaac aactaaaact ctgtgtttta gaatatcagg gcaagtatat tttaaaagtg
     1801 tgtggatgtg atgaatactt cctagaaaaa tatcctctga gtcagtataa gtatataaga
     1861 agctgtataa tgcttgggag gatgcccaat ttgatgttga tggctaaaga aagcctttat
     1921 tctcaactgc caatggactg ttttacaatg ccatcttatt ccagacgcat ttccacagct
     1981 acaccatata tgaatgggga aacatctaca aaatcccttt gggttataaa tagtgcactc
     2041 agaataaaaa ttctttgtgc aacctacgtg aatgtaaata ttcgagacat tgataagatc
     2101 tatgtccgaa caggtatcta ccatggagga gaacccttat gtgacaatgt gaacactcaa
     2161 agagtacctt gttccaatcc caggtggaat gaatggctga attatgatat atacattcct
     2221 gatcttcctc gtgctgctcg actttgcctt tccatttgct ctgttaaagg ccgaaagggt
     2281 gctaaagagg aacactgtcc attggcatgg ggaaatataa acttgtttga ttacacagac
     2341 actctagtat ctggaaaaat ggctttgaat ctttggccag tacctcatgg attagaagat
     2401 ttgctgaacc ctattggtgt tactggatca aatccaaata aagaaactcc atgcttagag
     2461 ttggagtttg actggttcag cagtgtggta aagttcccag atatgtcagt gattgaagag
     2521 catgccaatt ggtctgtatc ccgagaagca ggatttagct attcccacgc aggactgagt
     2581 aacagactag ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt
     2641 tctacacgag atcctctctc tgaaatcact aagcaggaga aagattttct atggagtcac
     2701 agacactatt gtgtaactat ccccgaaatt ctacccaaat tgcttctgtc tgttaaatgg
     2761 aattctagag atgaagtagc ccagatgtat tgcttggtaa aagattggcc tccaatcaaa
     2821 cctgaacagg ctatggaact tctggactgt aattacccag atcctatggt tcgaggtttt
     2881 gctgttcggt gcttggaaaa atatttaaca gatgacaaac tttctcagta tttaattcag
     2941 ctagtacagg tcctaaaata tgaacaatat ttggataact tgcttgtgag atttttactg
     3001 aagaaagcat tgactaatca aaggattggg cactttttct tttggcattt aaaatctgag
     3061 atgcacaata aaacagttag ccagaggttt ggcctgcttt tggagtccta ttgtcgtgca
     3121 tgtgggatgt atttgaagca cctgaatagg caagtcgagg caatggaaaa gctcattaac
     3181 ttaactgaca ttctcaaaca ggagaagaag gatgaaacac aaaaggtaca gatgaagttt
     3241 ttagttgagc aaatgaggcg accagatttc atggatgctc tacagggctt tctgtctcct
     3301 ctaaaccctg ctcatcaact aggaaacctc aggcttgaag agtgtcgaat tatgtcctct
     3361 gcaaaaaggc cactgtggtt gaattgggag aacccagaca tcatgtcaga gttactgttt
     3421 cagaacaatg agatcatctt taaaaatggg gatgatttac ggcaagatat gctaacactt
     3481 caaattattc gtattatgga aaatatctgg caaaatcaag gtcttgatct tcgaatgtta
     3541 ccttatggtt gtctgtcaat cggtgactgt gtgggactta ttgaggtggt gcgaaattct
     3601 cacactatta tgcaaattca gtgcaaaggc ggcttgaaag gtgcactgca gttcaacagc
     3661 cacacactac atcagtggct caaagacaag aacaaaggag aaatatatga tgcagccatt
     3721 gacctgttta cacgttcatg tgctggatac tgtgtagcta ccttcatttt gggaattgga
     3781 gatcgtcaca atagtaacat catggtgaaa gacgatggac aactgtttca tatagatttt
     3841 ggacactttt tggatcacaa gaagaaaaaa tttggttata aacgagaacg tgtgccattt
     3901 gttttgacac aggatttctt aatagtgatt agtaaaggag cccaagaatg cacaaagaca
     3961 agagaatttg agaggtttca ggagatgtgt tacaaggctt atctagctat tcgacagcat
     4021 gccaatctct tcataaatct tttctcaatg atgcttggct ctggaatgcc agaactacaa
     4081 tcttttgatg acattgcata cattcgaaag accctagcct tagataaaac tgagcaagag
     4141 gctttggagt atttcatgaa acaaatgaat gatgcacatc atggtggctg gacaacaaaa
     4201 atggattgga tcttccacac aattaaacag catgcattga actgataagg atcctgatga
     4261 gttaataaca ggcctgctgg taatcgcagg cctttttatt ttttttttta gatctagctt
     4321 catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat
     4381 attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa
     4441 tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag
     4501 tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga
     4561 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4621 tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat
     4681 tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag
     4741 ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat
     4801 ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag
     4861 ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact
     4921 gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg
     4981 gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa
     5041 gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat
     5101 ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     5161 tataagttgt agcggccgca cacagctaac accacgtcgt ccctatctgc tgccctaggt
     5221 ctatgagtgg ttgctggata actttacggg catgcataag gctcgtataa tatattcagg
     5281 gagaccacaa cggtttccct ctacaaataa ttttgtttaa ctttgaggga ctacaaaagt
     5341 acaagtaagg aggtatacat ggagcagaaa ttgattagcg aggaagattt gggtagcgac
     5401 ggtagcgccg gtccgaaagc taaaactacc acaccattgc aggtatgttt gttttaccct
     5461 aacctcattg gctacacacg cgttattctt tcattgattt ccttctgctt gctcggccac
     5521 tttcccattg catttttatt ttgttacatc attggttttg tgctggacgc ggtggatggc
     5581 atggtagcgc gacggtttgg ccaatgcaca cagtttggtg caatacttga tatgttgacg
     5641 gatcgggcgt caacggctgg tttgattgtt gtggttgtgc aggtattaca accgcttcca
     5701 cattggggag ccacttcgct tgcatgtctt gttttcctgg atatttcttc gcatttctgc
     5761 gtcatgtacg tgtcgcttta tgctggccgt actagtcata aggacgtgag cagcagtatt
     5821 ttttcacttc tgcgacttta ttatacaaat cgtccgttta tgtgtgcact ttgcgtcgga
     5881 caggaacttt tctacttaaa cttatatatg tacggcgtgt atgggatcgc tgaacctgcg
     5941 tttctgttct ttatggcttt gacagccgcg ttgagtgctt tcaagcaggt ggtgaatgtg
     6001 caacagctgc tggatagcat gtatcatctg gcggttctgg atgctgcggg aagccgccag
     6061 taatcgcact agccgctata ttaaggaggt aataaatggt ggaacaaaag ttgatcagcg
     6121 aagaagactt gggcagcgat ggtagcgcgg gtgcgtcggc ctcctccggg ccgtcgtctt
     6181 cggtcggttt ttcatccttt gatcccgcgg tcccttcctg taccttgtcc tcagcatctg
     6241 gaatcaagag acccatggca tctgaggtgc cttatgcctc tggcatgccc atcaagaaaa
     6301 taggccatag aagtgttgat tcctcaggag agacaacata taaaaagaca acctcatcag
     6361 ccttgaaagg tgccatccag ttaggcatta cccacactgt ggggagcctg agtaccaaac
     6421 cagagcgtga tgtcctcatg caagatttct acgtggttga gagtatcttc tttcccagtg
     6481 aagggagcaa cctgacccct gctcatcact acaatgactt tcgtttcaag acctatgcac
     6541 ctgttgcctt ccgctacttc cgggagctat ttggtatccg gcccgatgat tacttgtatt
     6601 ccctctgcag tgagccgctg attgaactct gtagctctgg agctagtggt tccctattct
     6661 atgtgtccag cgacgatgag ttcattatta agacagtcca acataaagag gcggaatttc
     6721 tgcagaagct gcttccagga tactacatga acctcaacca gaaccctcgg actttgctgc
     6781 ctaaattcta tggactgtac tgtgtgcagg caggtggcaa gaacattcgg attgtggtga
     6841 tgaacaatct tttaccaaga tcggtaaaaa tgcatatcaa atatgacctc aaaggctcaa
     6901 cctacaaacg gcgggcttcc cagaaagagc gagagaagcc tcttcccaca tttaaagacc
     6961 tagacttctt acaagacatc cctgatggtc tttttttgga tgctgacatg tacaacgctc
     7021 tctgtaagac cctgcagcgt gactgtttgg tgctgcagag cttcaagata atggattaca
     7081 gcctcttgat gtcaatccat aatatagatc atgcacaacg agagccctta agcagtgaaa
     7141 cacagtactc agttgatact cgaagaccgg ccccccaaaa ggctctgtat tccacagcca
     7201 tggaatccat ccagggagag gctcgacggg gtggtaccat ggagactgat gaccatatgg
     7261 gtggcatccc tgcccggaat agtaaagggg aaaggcttct gctttatatt ggcatcattg
     7321 acattctaca gtcttacagg tttgttaaga agttggagca ctcttggaaa gccctggtac
     7381 atgacggaga cactgtctca gtgcatcgcc caggcttcta cgctgaacgg ttccagcgct
     7441 tcatgtgcaa cacagtattt aagaagattc ccttgaagcc ttctccttcc aaaaagtttc
     7501 ggtctggctc atctttctct cggcgagcag gctccagtgg caactcctgc attacttacc
     7561 agccatcggt ctctggggaa cacaaggcac aagtgacaac aaaggcagaa gtggagccag
     7621 gcgttcacct tggtcgtcct gatgttttac ctcagactcc acctttggag gaaatcagtg
     7681 agggctcgcc tattcctgac cccagtttct cacctctagt tggagagact ttgcaaatgc
     7741 taactacaag tacaaccttg gaaaagcttg aagttgcaga gtcagagttc acccattaat
     7801 aataatgatg agttaataac agcctgctgg taatcgcagg cctttttatt ttttttttta
     7861 gctagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttattct
     7921 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7981 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     8041 agtatatatg agtaaacttg gtctgacag
//
