LOCUS       sequence-193963-        7163 bp    DNA     circular SYN 15-APR-2025
DEFINITION  This report is the first to directly measure nAChR subunit 
            stoichiometry using FRET and plasma membrane localization of 
            alpha-6- and beta-3-containing receptors using TIRF. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7163)
  AUTHORS   Drenan RM, Nashmi R, Imoukhuede P, Just H, McKinney S, Lester HA
  TITLE     Subcellular trafficking, pentameric assembly, and subunit 
            stoichiometry of neuronal nicotinic acetylcholine receptors 
            containing fluorescently labeled alpha6 and beta3 subunits.
  JOURNAL   Mol Pharmacol. 2008 Jan;73(1):27-41. Epub 2007 Oct 11.
  PUBMED    17932221
REFERENCE   2  (bases 1 to 7163)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol 
            Pharmacol."; date: "2008-01"; volume: "73(1)"; pages: "27-41. Epub 
            2007 Oct 11"
COMMENT     Sequence Label: pCDNA3.1-nAChr-beta3
FEATURES             Location/Qualifiers
     source          1..7163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        18..347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      198..333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     260..279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             396..1421
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             396..1421
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    1551..1672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1588..1607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1642..1661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1721..1737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1721..1737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1734..1756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1745..1761
                     /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(1769..1775,1776..1793,1794..1799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1814..1835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1952..1969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2123..2711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2203..2222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2882..3673,3674..3742))
                     /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     3505..3524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3743..3847)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3922..3941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4113..4492
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4493..4696
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4647..4667
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4741..4760
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4741..4759
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6463..6480)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6469..6693
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6739..7163,1..4)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6826..6845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7036..7057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      361 cttgtatatc cattttcgga tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga
      421 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
      481 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
      541 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
      601 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
      661 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
      721 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
      781 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
      841 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
      901 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
      961 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     1021 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     1081 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     1141 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     1201 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     1261 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     1321 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     1381 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtgcta cgagatttcg
     1441 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     1501 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     1561 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1621 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     1681 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     1741 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     1801 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     1861 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1921 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1981 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2041 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2101 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2161 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2221 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2281 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2341 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2401 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2461 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2521 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2581 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2641 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2701 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2761 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2821 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2881 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2941 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3001 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3061 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3121 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3181 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3241 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     3301 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3361 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3421 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3481 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3541 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3601 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3661 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3721 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3781 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3841 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     3901 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     3961 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     4021 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     4081 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
     4141 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     4201 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     4261 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     4321 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     4381 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     4441 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     4501 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     4561 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     4621 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     4681 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     4741 taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttggtacc
     4801 gagctcggat ccactagtcc agtgtggtgg aattctgcag atgaattcgc ccttcgtctc
     4861 tctgaagcag acgtcatgac aggcttccta cgggtcttct tggctctcag tgccactctc
     4921 tcaggttcct gggtgactct cacagccact gcgggactca gctcagtggc tgaacatgaa
     4981 gacgcactcc tcagacattt gttccaaggt tatcagaaat gtgtccgccc tgtgttgaat
     5041 tccagtgaca tcataaaagt gtattttgga ttaaagatat ctcagcttgt agatgtggat
     5101 gaaaagaatc agctgatgac aacaaacgtg tggctgaagc aggaatggac agaccaaaaa
     5161 ttacgatgga atcccgaaga ctatggtgga attaattcga taaaggttcc atcggagtcg
     5221 ctctggctgc ctgacatagt tctctttgaa aatgctgacg gacgttttga gggctctctc
     5281 atgaccaagg ccattgtgaa atccagcgga accgtcagtt ggactcctcc cgccagctac
     5341 aaaagttcct gtaccatgga tgtcacattt ttcccgttcg ataggcagaa ctgctcaatg
     5401 aagttcggat cctggactta cgatggtacc atggtcgacc tcattctgat caatgaaaac
     5461 gttgaccgga aagacttttt tgataacgga gagtgggaga tactaaatgc aaaggggatg
     5521 aagggcaaca gaagagaagg cttttactcc tatccgtttg ttacctactc ttttgttctg
     5581 agacgcctgc ccctgtttta caccctcttt ttgataatcc cctgcctggg gctgtctttt
     5641 ctcacggtcc tggtgttcta cctaccctcc gacgaagggg aaaaactctc cttatccacc
     5701 tcggttttgg tttctttgac agtgtttctt ttagtaatcg aagaaataat cccgtcttct
     5761 tcaaaagtca tccctctcat tggggagtac ctcctcttca ttatgatatt tgtcacgctg
     5821 tctattattg tcacggtttt tgtaattaat gtccaccaca gatcttcctc aacgtaccat
     5881 cccatggccc cctgggtaaa gaggctgttt ctggaaaaac tcccgagatg gctttgcatg
     5941 aaggacccca gggaccgctt ctctttcccc gatggaacgg agagtaaggg aaccgtgagg
     6001 gggaaatttc cagggaaaaa gaagcagact cctaccagtg atggagaaag agttctggtc
     6061 gctttcctgg agaaggcctc tgagtccatc agatacatct cgaggcatgt gaagaaggaa
     6121 cacttcatca gtcaggttgt tcaagactgg aaattcgtgg ctcaggttct tgaccgcatc
     6181 ttcctctggc tctttctgac agcttcggtt ttgggctctg tcctgatttt tattccagcc
     6241 ttgaagatgt ggatacatcg tttccattag gaactgctct ctggatccat gtagaagaca
     6301 cacctagaga gaatcccacc tagaactgag tgctgctggt gtgttcacag aaaggagttg
     6361 catgcaacat ttgtggtgcc cttgccttgt cctgggagcc aagggcgaat tccagcacac
     6421 tggcggccgt tactagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     6481 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     6541 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     6601 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     6661 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     6721 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6781 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     6841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     6961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     7021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     7081 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     7141 tgatttaaca aaaatttaac gcg
//
