LOCUS       sequence-311090-        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutant version of OlyA that senses sphingomyelin without or with 
            cholesterol.
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(E69A)
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             484..501
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             529..546
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(599..617)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      613..660
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      697..1152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(784..803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     994..1015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1178..1282
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1283..1351,1352..2143)
                     /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(1501..1520)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2314..2902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2803..2822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3056..3073
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3088..3227
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3153..3172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3313..3335
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3329..3520)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4329..5411)
                     /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"
     primer_bind     5373..5392
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5412..5489)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5695..5714
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5798..5816,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5798..5816
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tggcgtacgc ccaatgggtc atcattatca tccacaacgt cggatctcaa
      121 gacgtgaaga tcaagaacct caaggcttcc tgggggaagc ttcacgctga cggtgacaaa
      181 gacgctgaag tctcagcaag caactacgaa ggaaagatcg tcaagcctga cgagaagctc
      241 cagatcaacg cctctggcag atcagatgcc gccgcgggca cgaccggaac cttcgatctt
      301 gtcgaccctg ccgatggcga caagcaggtc cgccacttct actgggactc tccctggggc
      361 agcaaaacca acacttggac cgttagcggc tccaacacca agtggatgat cgagtactct
      421 gggcagaact tggacagcgg tgccctcggc accatcaccg tcgacacctt gaagaagggg
      481 aacctggttc cgcgcggcag cgaaggcaaa agcggctgcg gcctcgagca ccaccaccac
      541 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      601 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      661 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      721 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      781 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      841 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      901 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      961 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1021 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1081 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1141 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1201 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1261 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1321 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1381 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1441 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1501 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1561 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1621 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1681 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1741 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1801 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     1861 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1921 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1981 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     2041 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     2101 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     2161 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2221 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2281 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2341 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2401 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2461 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2521 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2581 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2641 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2701 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2761 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2821 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2881 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2941 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3001 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3061 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3121 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3181 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3241 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3301 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3361 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3421 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3481 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3541 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3601 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3661 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3721 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3781 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3841 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     3901 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     3961 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4021 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4081 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4141 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4201 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4261 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4441 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4501 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4561 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4621 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4681 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4741 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4801 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4861 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     4921 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     4981 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5041 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5101 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5161 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5221 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5281 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5341 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5401 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5461 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5521 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5581 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5641 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5701 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5761 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagg
//
