LOCUS       sequence-246740-        7899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Smoothened NQ4 in mammalian cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7899)
  AUTHORS   Marada S, Navarro G, Truong A, Stewart DP, Arensdorf AM, 
            Nachtergaele S, Angelats E, Opferman JT, Rohatgi R, McCormick PJ, 
            Ogden SK
  TITLE     Functional Divergence in the Role of N-Linked Glycosylation in 
            Smoothened Signaling.
  JOURNAL   PLoS Genet. 2015 Aug 20;11(8):e1005473. doi: 
            10.1371/journal.pgen.1005473. eCollection 2015 Aug.
  PUBMED    26291458
REFERENCE   2  (bases 1 to 7899)
  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"; journalName: "PLoS 
            Genet."; date: "2015-08-20"; volume: "11(8):e1005473. doi"; pages: "
            10.1371/journal.pgen.1005473. eCollection 2015 Aug"
COMMENT     Sequence Label: pCDNA3 mSmoothened NQ4
FEATURES             Location/Qualifiers
     source          1..7899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      713..722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3145..3154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3218..3236)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3219..3236)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3256..3273)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3262..3486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3532..3960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3619..3638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3829..3850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3969..3989)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3974..4303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4154..4289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4216..4235
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4370..5164
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4424..4443)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5034..5053
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5338..5459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5375..5394)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5429..5448
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5508..5524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5508..5524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5521..5543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5532..5548
                     /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(5556..5562,5563..5580,5581..5586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5601..5622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5739..5756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5910..6498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5990..6009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6669..7460,7461..7529))
                     /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     7292..7311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7530..7634)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7709..7728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattcg cggccgccat
      721 ggccgctggc cgccccgtgc gtgggcccga gctggcgccc cggaggctgc tgcagttgct
      781 tctgctggta ctgctggggg gcccgggccg gggggcggcc ttgagcgggc aagtgaccgg
      841 gcctgggcct cacagcgcca gcgggagctc gaggagggac gtgccggtga ccagccctcc
      901 gccgccgctg ctgagccact gcggccgggc cgcccactgc gagcctttgc gctacaacgt
      961 gtgcctgggc tcggcgctgc cctacggagc caccaccacg ctgctggctg gggactcgga
     1021 ctcgcaggag gaagcgcacg gcaagctcgt gctctggtcc ggcctccgga atgccccccg
     1081 ctgctgggca gtgatccagc ccctgctgtg tgctgtctac atgcccaagt gtgagaatga
     1141 ccgagtggag ttgcccagcc gtaccctctg ccaggccacc cgaggcccct gtgccattgt
     1201 ggagcgggag cgagggtggc ctgactttct gcgttgcaca ccggaccact tccctgaagg
     1261 ctgcccaaac gaggtacaaa acatcaagtt ccaaagctca ggccaatgtg aagcaccctt
     1321 ggtgcgaaca gacaacccca agagctggta tgaggacgtg gagggctgtg ggattcagtg
     1381 tcagaacccg ctgttcaccg aggccgagca ccaggacatg cacagctaca tcgcagcctt
     1441 cggggcggtc actggtctct gcacgctctt cacactggcc acctttgtgg ctgactggcg
     1501 gaactccaat cgctaccctg cggttattct cttctatgtc aatgcgtgtt tcttcgtggg
     1561 cagcattggc tggctggccc agttcatgga tggtgcccgc cgagagattg tttgccgagc
     1621 agatggcacc atgagatttg gggagcccac ctccagtgag accctgtcct gtgtcatcat
     1681 ctttgtcatt gtgtactatg ccttgatggc tggagtagtc tggttcgtgg tcctcaccta
     1741 tgcctggcac acctccttca aagccctggg caccacctac cagcctctct cgggcaagac
     1801 atcctatttc cacctgctca cgtggtcact cccctttgtc ctcacggtgg caatcctggc
     1861 tgtggctcag gtagatggag actccgtgag tggcatctgt tttgtaggct acaagaacta
     1921 tcggtaccgt gctggctttg tcctggcccc aattggcctg gtgcttattg tgggaggcta
     1981 cttcctcatc agaggggtca tgactctgtt ctccatcaag agcaaccacc ctgggcttct
     2041 gagtgagaag gcagccagca agatccaaga gaccatgctg cgcctgggca tttttggctt
     2101 cctggccttt ggctttgtgc tcatcacctt cagctgccac ttctatgact tcttcaacca
     2161 ggctgagtgg gagcgtagct tccgggacta tgtgctatgc caagcccaag tgaccatcgg
     2221 gctgcctacc aagaagccca ttcctgactg tgagatcaag aatcggccca gcctcctggt
     2281 ggagaagatc aatctatttg ccatgtttgg cactggcatt gccatgagca cctgggtctg
     2341 gaccaaggcc accctgctca tctggaggcg cacctggtgc aggttgactg ggcacagtga
     2401 tgatgagccc aagagaatca agaagagcaa gatgatcgcc aaggccttct ctaagcggcg
     2461 tgagctgctg cagaacccgg gccaggagct ctccttcagc atgcacactg tctcccatga
     2521 tggacctgtt gcgggtttgg cttttgacct caatgaaccc tcagctgacg tctcctctgc
     2581 ctgggctcag catgtcacca agatggtggc tcggagagga gccatattgc cccaggatgt
     2641 gtccgttacc cctgtggcaa ctccagtgcc accagaagaa caagccaaca tgtggctggt
     2701 tgaggcagag atctccccag agttagagaa gcgtttgggc cggaagaaaa agcggaggaa
     2761 gaggaagaag gaggtgtgcc ccttgaggcc agcccctgag cttcaccact ctgcccctgt
     2821 tcctgccacc agtgcagttc ctcggctgcc tcagctgcct cggcagaagt gcctggtagc
     2881 tgcaaacgcc tggggaacag gggagtcctg ccgacaggga gcctggactc tagtctccaa
     2941 ccccttctgc ccagagccta gtccccatca agatccattt ctccctggtg cctcagcccc
     3001 ccgggtctgg gctcagggcc gcctccaggg gctgggatcc attcattccc gcactaacct
     3061 aatggaggct gagatcttgg atgcagactc ggacttctga ggaattcctc agaagtccga
     3121 gtctgcatcc ggggcggcca gcggccatgg cggccgcgaa ttctgcagat atccatcaca
     3181 ctggcggccg ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta
     3241 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3301 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3361 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3421 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3481 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3541 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3601 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3661 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3721 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3781 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3841 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3901 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3961 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4081 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4141 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4201 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4261 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4321 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4381 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4441 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4501 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4561 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4621 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4681 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4741 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4801 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4861 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4921 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4981 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     5041 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5101 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5161 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5221 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5281 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5341 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5401 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5461 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5521 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5581 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5641 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5701 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5761 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5821 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5881 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5941 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6001 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6061 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6121 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6181 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6241 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6301 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6361 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6421 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgt
     6481 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6541 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6601 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6661 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6721 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6781 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6841 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6901 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6961 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7021 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7081 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7141 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7201 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7261 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7321 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7381 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7441 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7501 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7561 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7621 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7681 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7741 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     7801 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     7861 ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
