LOCUS       sequence-233237-        8087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ptch1-B (V111FL114FW115A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8087)
  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 8087)
  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-V111FL114FW115A-HA-GFP
FEATURES             Location/Qualifiers
     source          1..8087
                     /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"
     CDS             join(4846..4848,4849..5562)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4888..4909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5149..5168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5496..5517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(5578..5596)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5579..5596)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5616..5633)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5622..5846
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5930..5946)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5930..5946)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5943..5965)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5954..5970
                     /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(5978..5984,5985..6002,6003..6008))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6023..6044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6161..6178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6332..6920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6412..6431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7091..7882,7883..7951))
                     /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     7714..7733
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7952..8056)
                     /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 aacttcgagg agttcgcggt 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 acctcctagg 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 aggactgtcc 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 atgctgtgag caagggcgag
     4861 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     4921 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     4981 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     5041 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     5101 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     5161 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     5221 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     5281 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     5341 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     5401 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     5461 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     5521 gccgccggga tcactctcgg catggacgag ctgtacaagt agtctagagg gccctattct
     5581 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     5641 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     5701 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     5761 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     5821 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     5881 ggggtatccc cacgcgccct acctctagct agagcttggc gtaatcatgg tcatagctgt
     5941 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6001 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6061 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6121 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6181 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6241 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6301 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6361 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6421 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6481 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6541 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6601 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6661 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6721 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6781 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6841 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6901 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6961 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7021 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7081 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7141 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7201 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7261 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7321 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7381 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7441 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7501 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7561 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7621 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7681 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7741 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7801 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7861 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7921 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7981 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8041 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
