LOCUS       sequence-233160-        8086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ptch1-B for function assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8086)
  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 8086)
  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-HA-GFP
FEATURES             Location/Qualifiers
     source          1..8086
                     /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             4704..4817
                     /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             4818..4844
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(4845..4847,4848..5561)
                     /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(4887..4908)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5148..5167)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5495..5516
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(5577..5595)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5578..5595)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5615..5632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5621..5845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5929..5945)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5929..5945)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5942..5964)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5953..5969
                     /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(5977..5983,5984..6001,6002..6007))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6022..6043
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6160..6177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6331..6919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6411..6430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7090..7881,7882..7950))
                     /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     7713..7732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7951..8055)
                     /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 ggagacccag cttggtacca
      901 ccatggcctc ggctggtaac gccgccgggg ccctgggcag gcaggccggc ggcgggaggc
      961 gcagacggac cgggggaccg caccgcgccg cgccggaccg ggactatctg caccggccca
     1021 gctactgcga cgccgccttc gctctggagc agatttccaa ggggaaggct actggccgga
     1081 aagcgccgct gtggctgaga gcgaagtttc agagactctt atttaaactg ggttgttaca
     1141 ttcaaaagaa ctgcggcaag tttttggttg tgggtctcct catatttggg gccttcgctg
     1201 tgggattaaa ggcagctaat ctcgagacca acgtggagga gctgtgggtg gaagttggtg
     1261 gacgagtgag tcgagaatta aattataccc gtcagaagat aggagaagag gctatgttta
     1321 atcctcaact catgatacag actccaaaag aagaaggcgc taatgttctg accacagagg
     1381 ctctcctgca acacctggac tcagcactcc aggccagtcg tgtgcacgtc tacatgtata
     1441 acaggcaatg gaagttggaa catttgtgct acaaatcagg ggaacttatc acggagacag
     1501 gttacatgga tcagataata gaataccttt acccttgctt aatcattaca cctttggact
     1561 gcttctggga aggggcaaag ctacagtccg ggacagcata cctcctaggt aagcctcctt
     1621 tacggtggac aaactttgac cccttggaat tcctagaaga gttaaagaaa ataaactacc
     1681 aagtggacag ctgggaggaa atgctgaata aagccgaagt tggccatggg tacatggacc
     1741 ggccttgcct caacccagcc gacccagatt gccctgccac agcccctaac aaaaattcaa
     1801 ccaaacctct tgatgtggcc cttgttttga atggtggatg tcaaggttta tccaggaagt
     1861 atatgcattg gcaggaggag ttgattgtgg gtggtaccgt caagaatgcc actggaaaac
     1921 ttgtcagcgc tcacgccctg caaaccatgt tccagttaat gactcccaag caaatgtatg
     1981 aacacttcag gggctacgac tatgtctctc acatcaactg gaatgaagac agggcagccg
     2041 ccatcctgga ggcctggcag aggacttacg tggaggtggt tcatcaaagt gtcgccccaa
     2101 actccactca aaaggtgctt cccttcacaa ccacgaccct ggacgacatc ctaaaatcct
     2161 tctctgatgt cagtgtcatc cgagtggcca gcggctacct actgatgctt gcctatgcct
     2221 gtttaaccat gctgcgctgg gactgctcca agtcccaggg tgccgtgggg ctggctggcg
     2281 tcctgttggt tgcgctgtca gtggctgcag gattgggcct ctgctccttg attggcattt
     2341 cttttaatgc tgcgacaact caggttttgc cgtttcttgc tcttggtgtt ggtgtggatg
     2401 atgtcttcct cctggcccat gcattcagtg aaacaggaca gaataagagg attccatttg
     2461 aggacaggac tggggagtgc ctcaagcgca ccggagccag cgtggccctc acctccatca
     2521 gcaatgtcac cgccttcttc atggccgcat tgatccctat ccctgccctg cgagcgttct
     2581 ccctccaggc tgctgtggtg gtggtattca attttgctat ggttctgctc atttttcctg
     2641 caattctcag catggattta tacagacgtg aggacagaag attggatatt ttctgctgtt
     2701 tcacaagccc ctgtgtcagc agggtgattc aagttgagcc acaggcctac acagagccta
     2761 tgcagtccac cgttcagctc cgcacagagt atgaccctca cacgcacgtg tactacacca
     2821 ccgccgagcc acgctctgag atctctgtac agcctgttac cgtcacccag gacaacctca
     2881 gctgtcagag tcccgagagc accagctcta ccagggacct gctctcccag ttctcagact
     2941 ccagcctcca ctgcctcgag cccccctgca ccaagtggac actctcttcg tttgcagaga
     3001 agcactatgc tcctttcctc ctgaaaccca aagccaaggt tgtggtaatc cttcttttcc
     3061 tgggcttgct gggggtcagc ctttatggga ccacccgagt gagagacggg ctggacctca
     3121 cggacattgt tccccgggaa accagagaat atgacttcat agctgcccag ttcaagtact
     3181 tctctttcta caacatgtat atagtcaccc agaaagcaga ctacccgaat atccagcacc
     3241 tactttacga ccttcataag agtttcagca atgtgaagta tgtcatgctg gaggagaaca
     3301 agcaacttcc ccaaatgtgg ctgcactact ttagagactg gcttcaagga cttcaggatg
     3361 catttgacag tgactgggaa actgggagga tcatgccaaa caattataaa aatggatcag
     3421 atgacggggt cctcgcttac aaactcctgg tgcagactgg cagccgagac aagcccatcg
     3481 acattagtca gttgactaaa cagcgtctgg tagacgcaga tggcatcatt aatccgagcg
     3541 ctttctacat ctacctgacc gcttgggtca gcaacgaccc tgtagcttac gctgcctccc
     3601 aggccaacat ccggcctcac cggccggagt gggtccatga caaagccgac tacatgccag
     3661 agaccaggct gagaatccca gcagcagagc ccatcgagta cgctcagttc cctttctacc
     3721 tcaacggcct acgagacacc tcagactttg tggaagccat agaaaaagtg agagtcatct
     3781 gtaacaacta tacgagcctg ggactgtcca gctaccccaa tggctacccc ttcctgttct
     3841 gggagcaata catcagcctg cgccactggc tgctgctatc catcagcgtg gtgctggcct
     3901 gcacgtttct agtgtgcgca gtcttcctcc tgaacccctg gacggccggg atcattgtca
     3961 tggtcctggc tctgatgacc gttgagctct ttggcatgat gggcctcatt gggatcaagc
     4021 tgagtgctgt gcctgtggtc atcctgattg catctgttgg catcggagtg gagttcaccg
     4081 tccacgtggc tttggccttt ctgacagcca ttggggacaa gaaccacagg gctatgctcg
     4141 ctctggaaca catgtttgct cccgttctgg acggtgctgt gtccactctg ctgggtgtac
     4201 tgatgcttgc agggtccgaa tttgatttca ttgtcagata cttctttgcc gtcctggcca
     4261 ttctcaccgt cttgggggtt ctcaatggac tggttctgct gcctgtcctc ttatccttct
     4321 ttggaccgtg tcctgaggtg tctccagcca atggcctaaa ccgactgccc actccttcgc
     4381 ctgagccgcc tccaagtgtc gtccggtttg ccgtgcctcc tggtcacacg aacaatgggt
     4441 ctgattcctc cgactcggag tacagctctc agaccacggt gtctggcatc agtgaggagc
     4501 tcaggcaata cgaagcacag cagggtgccg gaggccctgc ccaccaagtg attgtggaag
     4561 ccacagaaaa ccctgtcttt gcccggtcca ctgtggtcca tccggactcc agacatcagc
     4621 ctcccttgac ccctcggcaa cagccccacc tggactctgg ctccttgtcc cctggacggc
     4681 aaggccagca gcctcgaagg gatatggatg aaaagaccac cggttggcgt ggtggtcatg
     4741 ttgttgaagg tctggcaggc gaactggaac agctgcgtgc acgtctggaa catcatccgc
     4801 agggtcagcg tgaaccgtat ccatatgatg ttccagatta tgctgtgagc aagggcgagg
     4861 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4921 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4981 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5041 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5101 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5161 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     5221 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     5281 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     5341 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5401 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     5461 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5521 ccgccgggat cactctcggc atggacgagc tgtacaagta gtctagaggg ccctattcta
     5581 tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     5641 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5701 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5761 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5821 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     5881 gggtatcccc acgcgcccta cctctagcta gagcttggcg taatcatggt catagctgtt
     5941 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6001 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6061 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6121 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6181 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6241 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6301 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6361 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6421 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6481 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6541 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6601 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6661 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6721 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6781 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6841 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6901 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6961 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7021 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7081 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7141 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7201 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7261 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7321 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7381 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7441 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7501 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7561 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7621 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7681 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7741 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7801 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7861 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7921 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7981 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8041 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
