LOCUS       sequence-233158-        7373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ptch1-B  (Y233C) for function assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   Zhang Y, Bulkley DP, Xin Y, Roberts KJ, Asarnow DE, Sharma A, Myers 
            BR, Cho W, Cheng Y, Beachy PA
  TITLE     Structural Basis for Cholesterol Transport-like Activity of the 
            Hedgehog Receptor Patched.
  JOURNAL   Cell. 2018 Nov 15;175(5):1352-1364.e14. doi: 
            10.1016/j.cell.2018.10.026. Epub 2018 Nov 8.
  PUBMED    30415841
REFERENCE   2  (bases 1 to 7373)
  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.2018.10.026"; journalName: "Cell"; date: 
            "2018-11-15- 15"; volume: "175"; issue: "5"; pages: "1352-1364.e14"
COMMENT     Sequence Label: pcDNA-h-mmPtch1-B-Y233C-HA
FEATURES             Location/Qualifiers
     source          1..7373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4705..4818
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             4819..4845
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(4864..4882)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4865..4882)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4902..4919)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4908..5132
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5216..5232)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5216..5232)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5229..5251)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5240..5256
                     /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)."
     promoter        complement(join(5264..5270,5271..5288,5289..5294))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5309..5330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5447..5464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5618..6206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5698..5717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6377..7168,7169..7237))
                     /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     7000..7019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7238..7342)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg gtactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 accatggcct cggctggtaa cgccgccggg gccctgggca ggcaggccgg cggcgggagg
      961 cgcagacgga ccgggggacc gcaccgcgcc gcgccggacc gggactatct gcaccggccc
     1021 agctactgcg acgccgcctt cgctctggag cagatttcca aggggaaggc tactggccgg
     1081 aaagcgccgc tgtggctgag agcgaagttt cagagactct tatttaaact gggttgttac
     1141 attcaaaaga actgcggcaa gtttttggtt gtgggtctcc tcatatttgg ggccttcgct
     1201 gtgggattaa aggcagctaa tctcgagacc aacgtggagg agctgtgggt ggaagttggt
     1261 ggacgagtga gtcgagaatt aaattatacc cgtcagaaga taggagaaga ggctatgttt
     1321 aatcctcaac tcatgataca gactccaaaa gaagaaggcg ctaatgttct gaccacagag
     1381 gctctcctgc aacacctgga ctcagcactc caggccagtc gtgtgcacgt ctacatgtat
     1441 aacaggcaat ggaagttgga acatttgtgc tacaaatcag gggaacttat cacggagaca
     1501 ggttacatgg atcagataat agaatacctt tacccttgct taatcattac acctttggac
     1561 tgcttctggg aaggggcaaa gctacagtcc gggacagcat gcctcctagg taagcctcct
     1621 ttacggtgga caaactttga ccccttggaa ttcctagaag agttaaagaa aataaactac
     1681 caagtggaca gctgggagga aatgctgaat aaagccgaag ttggccatgg gtacatggac
     1741 cggccttgcc tcaacccagc cgacccagat tgccctgcca cagcccctaa caaaaattca
     1801 accaaacctc ttgatgtggc ccttgttttg aatggtggat gtcaaggttt atccaggaag
     1861 tatatgcatt ggcaggagga gttgattgtg ggtggtaccg tcaagaatgc cactggaaaa
     1921 cttgtcagcg ctcacgccct gcaaaccatg ttccagttaa tgactcccaa gcaaatgtat
     1981 gaacacttca ggggctacga ctatgtctct cacatcaact ggaatgaaga cagggcagcc
     2041 gccatcctgg aggcctggca gaggacttac gtggaggtgg ttcatcaaag tgtcgcccca
     2101 aactccactc aaaaggtgct tcccttcaca accacgaccc tggacgacat cctaaaatcc
     2161 ttctctgatg tcagtgtcat ccgagtggcc agcggctacc tactgatgct tgcctatgcc
     2221 tgtttaacca tgctgcgctg ggactgctcc aagtcccagg gtgccgtggg gctggctggc
     2281 gtcctgttgg ttgcgctgtc agtggctgca ggattgggcc tctgctcctt gattggcatt
     2341 tcttttaatg ctgcgacaac tcaggttttg ccgtttcttg ctcttggtgt tggtgtggat
     2401 gatgtcttcc tcctggccca tgcattcagt gaaacaggac agaataagag gattccattt
     2461 gaggacagga ctggggagtg cctcaagcgc accggagcca gcgtggccct cacctccatc
     2521 agcaatgtca ccgccttctt catggccgca ttgatcccta tccctgccct gcgagcgttc
     2581 tccctccagg ctgctgtggt ggtggtattc aattttgcta tggttctgct catttttcct
     2641 gcaattctca gcatggattt atacagacgt gaggacagaa gattggatat tttctgctgt
     2701 ttcacaagcc cctgtgtcag cagggtgatt caagttgagc cacaggccta cacagagcct
     2761 atgcagtcca ccgttcagct ccgcacagag tatgaccctc acacgcacgt gtactacacc
     2821 accgccgagc cacgctctga gatctctgta cagcctgtta ccgtcaccca ggacaacctc
     2881 agctgtcaga gtcccgagag caccagctct accagggacc tgctctccca gttctcagac
     2941 tccagcctcc actgcctcga gcccccctgc accaagtgga cactctcttc gtttgcagag
     3001 aagcactatg ctcctttcct cctgaaaccc aaagccaagg ttgtggtaat ccttcttttc
     3061 ctgggcttgc tgggggtcag cctttatggg accacccgag tgagagacgg gctggacctc
     3121 acggacattg ttccccggga aaccagagaa tatgacttca tagctgccca gttcaagtac
     3181 ttctctttct acaacatgta tatagtcacc cagaaagcag actacccgaa tatccagcac
     3241 ctactttacg accttcataa gagtttcagc aatgtgaagt atgtcatgct ggaggagaac
     3301 aagcaacttc cccaaatgtg gctgcactac tttagagact ggcttcaagg acttcaggat
     3361 gcatttgaca gtgactggga aactgggagg atcatgccaa acaattataa aaatggatca
     3421 gatgacgggg tcctcgctta caaactcctg gtgcagactg gcagccgaga caagcccatc
     3481 gacattagtc agttgactaa acagcgtctg gtagacgcag atggcatcat taatccgagc
     3541 gctttctaca tctacctgac cgcttgggtc agcaacgacc ctgtagctta cgctgcctcc
     3601 caggccaaca tccggcctca ccggccggag tgggtccatg acaaagccga ctacatgcca
     3661 gagaccaggc tgagaatccc agcagcagag cccatcgagt acgctcagtt ccctttctac
     3721 ctcaacggcc tacgagacac ctcagacttt gtggaagcca tagaaaaagt gagagtcatc
     3781 tgtaacaact atacgagcct gggactgtcc agctacccca atggctaccc cttcctgttc
     3841 tgggagcaat acatcagcct gcgccactgg ctgctgctat ccatcagcgt ggtgctggcc
     3901 tgcacgtttc tagtgtgcgc agtcttcctc ctgaacccct ggacggccgg gatcattgtc
     3961 atggtcctgg ctctgatgac cgttgagctc tttggcatga tgggcctcat tgggatcaag
     4021 ctgagtgctg tgcctgtggt catcctgatt gcatctgttg gcatcggagt ggagttcacc
     4081 gtccacgtgg ctttggcctt tctgacagcc attggggaca agaaccacag ggctatgctc
     4141 gctctggaac acatgtttgc tcccgttctg gacggtgctg tgtccactct gctgggtgta
     4201 ctgatgcttg cagggtccga atttgatttc attgtcagat acttctttgc cgtcctggcc
     4261 attctcaccg tcttgggggt tctcaatgga ctggttctgc tgcctgtcct cttatccttc
     4321 tttggaccgt gtcctgaggt gtctccagcc aatggcctaa accgactgcc cactccttcg
     4381 cctgagccgc ctccaagtgt cgtccggttt gccgtgcctc ctggtcacac gaacaatggg
     4441 tctgattcct ccgactcgga gtacagctct cagaccacgg tgtctggcat cagtgaggag
     4501 ctcaggcaat acgaagcaca gcagggtgcc ggaggccctg cccaccaagt gattgtggaa
     4561 gccacagaaa accctgtctt tgcccggtcc actgtggtcc atccggactc cagacatcag
     4621 cctcccttga cccctcggca acagccccac ctggactctg gctccttgtc ccctggacgg
     4681 caaggccagc agcctcgaag ggatatggat gaaaagacca ccggttggcg tggtggtcat
     4741 gttgttgaag gtctggcagg cgaactggaa cagctgcgtg cacgtctgga acatcatccg
     4801 cagggtcagc gtgaaccgta tccatatgat gttccagatt atgcttagtc tagagggccc
     4861 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     4921 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4981 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     5041 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5101 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     5161 ctctaggggg tatccccacg cgccctacct ctagctagag cttggcgtaa tcatggtcat
     5221 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5281 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5341 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5401 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5461 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5521 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5581 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5641 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5701 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5761 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5821 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5881 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5941 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6001 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6061 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6121 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6181 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6241 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6301 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6361 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6421 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6481 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6541 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6601 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6661 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6721 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6781 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6841 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6901 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6961 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7021 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7081 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7141 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7201 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7321 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
