LOCUS       sequence-234207-        8111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mmPtch1-B NNQ variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8111)
  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 8111)
  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-NNQ-HA-GFP
FEATURES             Location/Qualifiers
     source          1..8111
                     /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..4725
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4729..4842
                     /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             4843..4869
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(4870..4872,4873..5586)
                     /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(4912..4933)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5173..5192)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5520..5541
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(5602..5620)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5603..5620)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5640..5657)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5646..5870
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5954..5970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5954..5970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5967..5989)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5978..5994
                     /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(6002..6008,6009..6026,6027..6032))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6047..6068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6185..6202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6356..6944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6436..6455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7115..7906,7907..7975))
                     /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     7738..7757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7976..8080)
                     /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 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 tggtgtgaat
     2401 aatgtcttcc 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 gcagttcacc
     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 ggatgaaaat ctgtacttcc agtccggcat ggatgaaaag
     4741 accaccggtt ggcgtggtgg tcatgttgtt gaaggtctgg caggcgaact ggaacagctg
     4801 cgtgcacgtc tggaacatca tccgcagggt cagcgtgaac cgtatccata tgatgttcca
     4861 gattatgctg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4921 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     4981 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     5041 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     5101 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     5161 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     5221 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     5281 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     5341 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     5401 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     5461 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     5521 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     5581 aagtagtcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     5641 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     5701 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     5761 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     5821 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     5881 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctacctct agctagagct
     5941 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6001 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6061 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6121 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6181 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6241 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6301 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6361 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6421 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6481 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6541 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6601 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6661 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6721 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6781 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6841 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6901 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6961 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7021 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7981 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8101 cacctgacgt c
//
