LOCUS       sequence-311089-        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ostreolysin A for sensing sphingomyelin/cholesterol complexes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5816)
  AUTHORS   Endapally S, Frias D, Grzemska M, Gay A, Tomchick DR, Radhakrishnan 
            A
  TITLE     Molecular Discrimination between Two Conformations of Sphingomyelin 
            in Plasma Membranes.
  JOURNAL   Cell. 2019 Feb 21;176(5):1040-1053.e17. doi: 
            10.1016/j.cell.2018.12.042. Epub 2019 Jan 31.
  PUBMED    30712872
REFERENCE   2  (bases 1 to 5816)
  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.cell.2018.12.042"; journalName: "Cell"; date: 
            "2019-02-21- 21"; volume: "176"; issue: "5"; pages: "1040-1053.e17"
COMMENT     Sequence Label: pOlyA(WT)
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1490..1507
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1535..1552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1605..1623)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1619..1666
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1703..2158
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1790..1809)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2000..2021
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2184..2288
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2289..2357,2358..3149)
                     /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(2507..2526)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3320..3908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3809..3828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4062..4079
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4094..4233
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4159..4178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4319..4341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4335..4526)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5335..5816,1..601))
                     /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 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatggc
     1081 gtacgcccaa tgggtcatca ttatcatcca caacgtcgga tctcaagacg tgaagatcaa
     1141 gaacctcaag gcttcctggg ggaagcttca cgctgacggt gacaaagacg ctgaagtctc
     1201 agcaagcaac tacgaaggaa agatcgtcaa gcctgacgag aagctccaga tcaacgcctc
     1261 tggcagatca gatgccgccg aaggcacgac cggaaccttc gatcttgtcg accctgccga
     1321 tggcgacaag caggtccgcc acttctactg ggactctccc tggggcagca aaaccaacac
     1381 ttggaccgtt agcggctcca acaccaagtg gatgatcgag tactctgggc agaacttgga
     1441 cagcggtgcc ctcggcacca tcaccgtcga caccttgaag aaggggaacc tggttccgcg
     1501 cggcagcgaa ggcaaaagcg gctgcggcct cgagcaccac caccaccacc actgagatcc
     1561 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1621 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1681 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1741 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1801 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1861 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1921 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1981 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2041 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2101 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2161 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2221 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2281 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2341 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2401 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2461 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2521 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2581 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2641 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2701 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2761 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2821 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2881 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2941 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3001 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3061 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3121 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3181 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3241 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3301 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3361 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3421 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3481 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3541 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3601 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3661 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3721 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3781 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3841 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3901 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3961 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4021 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4081 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4141 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4201 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4261 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4321 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4381 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4441 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4501 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4561 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4621 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4681 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4741 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4801 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4861 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4921 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4981 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5041 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5101 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5161 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5221 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5281 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5341 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5401 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5461 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5521 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5581 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     5641 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5701 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     5761 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgcc
//
