LOCUS       sequence-360706-        8587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  alpha7 nACHR expression in mammalian cell lines with calcium 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8587)
  AUTHORS   Belanger-Coast MG, Zhang M, Bugay V, Gutierrez RA, Gregory SR, Yu W,
            Brenner R
  TITLE     Dequalinium chloride is an antagonists of alpha7 nicotinic 
            acetylcholine receptors.
  JOURNAL   Eur J Pharmacol. 2022 Jun 15;925:175000. doi: 
            10.1016/j.ejphar.2022.175000. Epub 2022 May 4.
  PUBMED    35525312
REFERENCE   2  (bases 1 to 8587)
  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: "Eur J 
            Pharmacol."; date: "2022-06-15"; volume: "925:175000. doi"; pages: "
            10.1016/j.ejphar.2022.175000. Epub 2022 May 4"
COMMENT     Sequence Label: alpha7nACHR_Nacho_GCAMP7s
FEATURES             Location/Qualifiers
     source          1..8587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        22..1200
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          253..1191
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1148..1168
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1200..1224
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2740..2764)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2765..3339,3340..3342,3343..3351)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2932..2949)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3159..3178
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3871..3900
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3919..3942
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(3949..3969)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3997..4118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4008..4027)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4062..4081
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        4216..4595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4596..4799
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4750..4770
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4847..4856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4853..4861,4862..4879,4880..4882,4883..4915,4916..4918,
                     4919..4942,4943..4969,4970..5026,5027..5032,5033..5302,
                     5303..5320,5321..5755,5756..5761,5762..6202)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRVIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      4949..4958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5239..5260
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5366..5387)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5627..5646)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(6326..6343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6332..6556
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6581..6598)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6752..7340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6832..6851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7511..8302,8303..8371))
                     /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     8134..8153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8372..8476)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8544..8562
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 caactttgta tagaaaagtt gggctccggt gcccgtcagt gggcagagcg cacatcgccc
       61 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
      121 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
      181 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
      241 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat
      301 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag
      361 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct
      421 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac
      481 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct
      541 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact
      601 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt
      661 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct
      721 ggccggcctg ctctggtgcc tggtctcgcg ccgccgtgta tcgccccgcc ctgggcggca
      781 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct
      841 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca
      901 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg
      961 gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
     1021 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg
     1081 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg
     1141 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga
     1201 caagtttgta caaaaaagca ggctgccacc atgcgctgct cgccgggagg cgtctggctg
     1261 gcgctggccg cgtcgctcct gcacgtgtcc ctgcaaggcg agttccagag gaagctttac
     1321 aaggagctgg tcaagaacta caatcccctg gagaggcccg tggccaatga ctcgcaacca
     1381 ctcaccgtct acttctccct gagcctcctg cagatcatgg acgtggatga gaagaaccaa
     1441 gttttaacca ccaacatttg gctgcaaatg tcttggacag atcactattt acagtggaat
     1501 gtgtcagaat atccaggggt gaagactgtt cgtttcccag atggccagat ttggaaacca
     1561 gacattcttc tctataacag tgctgatgag cgctttgacg ccacattcca cactaacgtg
     1621 ttggtgaatt cttctgggca ttgccagtac ctgcctccag gcatattcaa gagttcctgc
     1681 tacatcgatg tacgctggtt tccctttgat gtgcagcact gcaaactgaa gtttgggtcc
     1741 tggtcttacg gaggctggtc cttggatctg cagatgcagg aggcagatat cagtggctat
     1801 atccccaatg gagaatggga cctagtggga atccccggca agaggagtga aaggttctat
     1861 gagtgctgca aagagcccta ccctgatgtc accttcacag tgaccatgcg ccgcaggaca
     1921 ctctactatg gcctcaacct gctgatcccc tgtgtgctca tctccgccct cgccctgctg
     1981 gtgttcctgc ttcctgcaga ttccggggag aagatttccc tggggataac agtcttactc
     2041 tctcttaccg tcttcatgct gctcgtggct gagatcatgc ccgcaacatc cgattcggta
     2101 ccattgatag cccagtactt cgccagcacc atgatcatcg tgggcctctc ggtggtggtg
     2161 acggtgatcg tgctgcagta ccaccaccac gaccccgacg ggggcaagat gcccaagtgg
     2221 accagagtca tccttctgaa ctggtgcgcg tggttcctgc gaatgaagag gcccggggag
     2281 gacaaggtgc gcccggcctg ccagcacaag cagcggcgct gcagcctggc cagtgtggag
     2341 atgagcgccg tggcgccgcc gcccgccagc aacgggaacc tgctgtacat cggcttccgc
     2401 ggcctggacg gcgtgcactg tgtcccgacc cccgactctg gggtagtgtg tggccgcatg
     2461 gcctgctccc ccacgcacga tgagcacctc ctgcacggtg ggcaaccccc cgagggggac
     2521 ccggacttgg ccaagatcct ggaggaggtc cgctacattg ccaaccgctt ccgctgccag
     2581 gacgaaagcg aggcggtctg cagcgagtgg aagttcgccg cctgtgtggt ggaccgcctg
     2641 tgcctcatgg ccttctcggt cttcaccatc atctgcacca tcggcatcct gatgtcggct
     2701 cccaacttcg tggaggccgt gtccaaagac tttgcgtaaa cccagctttc ttgtacaaag
     2761 tgggcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2821 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2881 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2941 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     3001 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     3061 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     3121 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     3181 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3241 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3301 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggcatcc
     3361 ccacgaacca taactatcat ggccctctca gtggccctgg gactcttctt tgttttcatg
     3421 gggaccatca agttgacccc caggctcagc aaggatgcct acagtgaaat gaaacgtgct
     3481 tacaagagct acgttcgagc ccttcctctg ctgaagaaaa tgggcatcaa ttccatcctc
     3541 cttcggaaaa gtattggtgc tcttgaagtg gcctgtggca ttgtcatgac cctggtaccc
     3601 ggtcgtccca aagatgtggc caacttcttc ctgctcttgc tggtgttggc tgtgctcttc
     3661 ttccaccagc tggtgggtga tcctctcaaa cgctacgccc atgctctggt gtttggaatc
     3721 ctgctcacct gccgcctgct gattgcccgc aagcctgaag acagatcttc tgagaagaag
     3781 gccttgcctg agagtgcaga ggagcaacca tccttgtatg agaaggcccc ccagggcaaa
     3841 gtgaaggtgt caacgcgtac gcggccgctc gagcagaaac tcatctcaga agaggatctg
     3901 gcagcaaatg atatcctgga ttacaaggat gacgacgata aggtttaaca actttattat
     3961 acatagttga tggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     4021 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     4081 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     4141 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     4201 aatgtggtac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     4261 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     4321 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     4381 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     4441 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     4501 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     4561 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     4621 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     4681 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     4741 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     4801 gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgcca ccatgggttc
     4861 tcatcatcat catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga
     4921 tctgtacgac gatgacgata aggatctcgc caccatggtc gactcatcac gtcgtaagtg
     4981 gaataagaca ggtcacgcag tcagagtgat aggtcggctg agctcactcg agaacgtcta
     5041 tatcaaggcc gacaagcaga agaacggcat caaggcgaac ttccacatcc gccacaacat
     5101 cgaggacggc ggcgtgcagc tcgcctacca ctaccagcag aacaccccca tcggcgacgg
     5161 ccccgtgctg ctgcccgaca accactacct gagcgtgcag tccaaacttt cgaaagaccc
     5221 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     5281 cggcatggac gagctgtaca agggcggtac cggagggagc atggtgagca agggcgagga
     5341 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     5401 gttcagcgtg tccggcgagg gtgagggcga tgccacctac ggcaagctga ccctgaagtt
     5461 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     5521 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5581 cgccatgccc gaaggctaca tccaggagcg caccatcttc ttcaaggacg acggcaacta
     5641 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagcttaa
     5701 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaaccttcc
     5761 tgaccaactg actgaagagc agatcgcaga atttaaagag cttttctccc tatttgacaa
     5821 ggacggggat gggacaataa caaccaagga gctggggacg gtgatgcggt ctctggggca
     5881 gaaccccaca gaagcagagc tgcaggacat gatcaatgaa gtagatgccg acggtgacgg
     5941 cacaatcgac ttccctgagt tcctgacaat gatggcaaga aaaatgaaat acagggacac
     6001 ggaagaagaa attagagaag cgttcggtgt gtttgataag gatggcaatg gctacatcag
     6061 tgcagcagag cttcgccacg tgatgacaaa ccttggagag aagttaacag atgaagaggt
     6121 tgatgaaatg atcagggaag cagacatcga tggggatggt caggtaaact acgaagagtt
     6181 tgtacaaatg atgacagcga agggagcagg agcaaaagaa aagatgagca aagatggtaa
     6241 aaagaagaaa aagaagtcaa agacaaagtg tgtaattatg taagcggccg cgactctaga
     6301 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6361 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6421 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     6481 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     6541 gcggtgggct ctatgggcgg ccgcggcgct cttccgcttc ctcgctcact gactcgctgc
     6601 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6661 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6721 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6781 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6841 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6901 gatacctgtc cgcctttctc tcttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6961 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     7021 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7081 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7141 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     7201 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7261 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7321 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7381 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7441 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7501 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7561 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7621 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7681 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7741 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7801 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7861 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7921 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7981 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     8041 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8101 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8161 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8221 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8281 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8341 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8401 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8461 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     8521 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cggcgcgccg
     8581 cggccgc
//
