LOCUS       sequence-214044-        3190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses domain 4 of anthrolysin O cholesterol sensor, has 
            N-terminal His6-tag and two point mutations, S404C and C472A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3190)
  AUTHORS   Gay A, Rye D, Radhakrishnan A
  TITLE     Switch-like responses of two cholesterol sensors do not require 
            protein oligomerization in membranes.
  JOURNAL   Biophys J. 2015 Mar 24;108(6):1459-69. doi: 
            10.1016/j.bpj.2015.02.008.
  PUBMED    25809258
REFERENCE   2  (bases 1 to 3190)
  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.bpj.2015.02"; journalName: "Biophys J"; date: 
            "2015-03-24- 24"; volume: "108"; issue: "6"; pages: "1459-69"
COMMENT     Sequence Label: pALOD4
FEATURES             Location/Qualifiers
     source          1..3190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(591..609)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      605..652
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      749..1204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(836..855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1046..1067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1230..1334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1335..1403,1404..2195)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1553..1572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2366..2954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2855..2874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3108..3125
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatccgtg cgctaaaatg accctggatc actatggcgc atacgtcgct
      241 cagtttgatg ttagttggga cgaatttacc ttcgatcaaa acggcaagga agttctgacc
      301 cataaaacgt gggaaggcag tggtaaagat aagaccgcgc actattccac ggttatcccg
      361 ctgccgccga actcaaaaaa tattaagatc gtcgcacgtg aagcgaccgg tctggcatgg
      421 gaatggtggc gtacgattat caacgaacag aatgtgccgc tgaccaatga aatcaaggtc
      481 tctattggcg gcaccacgct gtatccgacg gcaaccatct cacactaaga attcgaagct
      541 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
      601 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
      661 aggaactata tccggatctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      721 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
      781 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      841 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      901 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      961 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1021 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1081 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1141 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1201 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3121 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3181 attaatgcag
//
