LOCUS       sequence-193964-        7178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of mouse Alpha6 (Chrna6) with internal mEGFP 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7178)
  AUTHORS   Xiao C, Srinivasan R, Drenan RM, Mackey ED, McIntosh JM, Lester HA
  TITLE     Characterizing functional alpha6beta2 nicotinic acetylcholine 
            receptors in vitro: mutant beta2 subunits improve membrane 
            expression, and fluorescent proteins reveal responsive cells.
  JOURNAL   Biochem Pharmacol. 2011 Oct 15;82(8):852-61. Epub 2011 May 17.
  PUBMED    21609715
REFERENCE   2  (bases 1 to 7178)
  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: "Biochem 
            Pharmacol."; date: "2011-10-15"; volume: "82(8)"; pages: "852-61. 
            Epub 2011 May 17"
COMMENT     Sequence Label: pcDNA-zeo: alpha6mGFP
FEATURES             Location/Qualifiers
     source          1..7178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1774..1793)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1775..1793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1838..2041)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1867..1887)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2042..2421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2593..2612
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2687..2791
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2792..2860,2861..3652)
                     /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     complement(3010..3029)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3823..4411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4312..4331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4565..4582
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4699..4720
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4735..4740,4741..4758,4759..4765)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4773..4789
                     /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)."
     primer_bind     4778..4800
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4797..4813
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4797..4813
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4862..4983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4873..4892)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4927..4946
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5113..5487)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(5506..5553)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5601..5930)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5615..5750
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5669..5688)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5915..5935
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5944..6372)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6054..6075)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6266..6285
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6418..6642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6631..6648
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6965..7178,1..497,498..500,501..503))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7007..7028)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      301 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcac cattgctcct gctgctatgt cacttgattt ttggcagtgg
      541 tggcattcct ttagaagttt ggattctcct cgatgagtaa atttgacctt ggcaggcctc
      601 ttggtatggc ttttggggtc cttaacacct cctgcctcct ttgtcttgtc cagaggtttc
      661 ctcatcatca gaatcgaggg gaaggcctga aggaagatgg tcttcaccca cttgggcatg
      721 gtatgcgttg ctggggtcct gtagtgtatg ttcagcacga acacggtcac cacaatggac
      781 agcgtgacaa agatcatggt gaacagtaga tactcaccca ccagtgggat cacgagagat
      841 gtggatggga tggtctctgt aatcaccagc aaaaagacag tgagagaaag cagaacggag
      901 atgcaaagag tcactttctc gccacagtcg gagggaaggt aaaaaaccag caccgtgagg
      961 aaggaaatga agaggcaggg gatgatgagg ttgatggtgt aaaacatggg caacctcctg
     1021 atgtagaagg agtaggttat gtccgtgtaa atctcttcac agcagttgta cttgatgtca
     1081 tgcttatagc cagaggcgtc gacaatttcc cattcactgt tttcccaaaa gtcgttcatg
     1141 tctactttag agccaatgat gagaaggtcg atttctgcct tgtcataagt ccaggaacca
     1201 aacttcaggg agcagttttg atggtcaaac gggaagaagg tgatgtccat tgggcaggag
     1261 ctcttaaaga tggctggtgg ggtccaggtt atcacaccgt catacttgag aagagctttg
     1321 gtcttgcctt cgacctggaa gtcgccgaca gcattgttat acagaacgat gtcaggcttc
     1381 cagatgttgt ctgctggaac tcgaagtgtc tcgatgccat catactccgt tggatcccaa
     1441 cgcaatctgt agtccttcca gatgtgacgc agccacagat tggtctccat aatctggttg
     1501 acttcatcca cattggccag ttgcgtgatt gccaattcaa aatgcaccgt gacgggatcg
     1561 gagacattct ccaccggccg gatgaagcgg ttgtagtgag caaacagcct gtggaagagc
     1621 tgctcttcag attcacagcc tgtactgcct ttaaagagag ccaggaatcc acatagccac
     1681 agacataaac cggagtgcag gtttccctgg tcccggctgt tcagcatggt ggcggtacca
     1741 agcttaagtt taaacgctag ccagcttggg tctccctata gtgagtcgta ttaatttcga
     1801 taagccagta agcagtgggt tctctagtta gccagagagc tctgcttata tagacctccc
     1861 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
     1921 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
     1981 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
     2041 ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
     2101 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     2161 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     2221 cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg
     2281 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     2341 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
     2401 aactagtcaa taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa
     2461 aacgcctaac cctaagcaga ttcttcatgc aattgtcggt caagccttgc cttgttgtag
     2521 cttaaatttt gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact
     2581 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag
     2641 atctcccgat ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2701 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2761 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2821 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2881 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2941 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3001 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3061 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3121 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3181 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3241 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3301 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3361 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3421 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3481 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3541 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3601 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3661 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3721 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3781 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3841 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3901 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3961 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4021 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4081 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4141 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4201 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4261 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4321 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4381 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4441 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4501 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4561 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     4621 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     4681 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     4741 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     4801 aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg
     4861 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     4921 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     4981 gttggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc
     5041 gtccaggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc
     5101 tcgtagcacg tgtcagtcct gctcctcggc cacgaagtgc acgcagttgc cggccgggtc
     5161 gcgcagggcg aactcccgcc cccacggctg ctcgccgatc tcggtcatgg ccggcccgga
     5221 ggcgtcccgg aagttcgtgg acacgacctc cgaccactcg gcgtacagct cgtccaggcc
     5281 gcgcacccac acccaggcca gggtgttgtc cggcaccacc tggtcctgga ccgcgctgat
     5341 gaacagggtc acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga agtcccggga
     5401 gaacccgagc cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg cggtgagcac
     5461 cggaacggca ctggtcaact tggccatggt ttagttcctc accttgtcgt attatactat
     5521 gccgatatac tatgccgatg attaattgtc aacacgtgct gatcagatcc gaaaatggat
     5581 atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac
     5641 ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt
     5701 cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt
     5761 aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     5821 ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata
     5881 cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac attccacaga
     5941 attaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6001 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     6061 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6121 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6181 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6241 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6301 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6361 tacagggcgc gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca
     6421 tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt
     6481 cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt
     6541 cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg
     6601 gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg
     6661 tttaaacggg ccctctagac tcgagttaag actttcctgt gttcccaagc agtggctgca
     6721 gaaatagccc cacagttcca aacacacaga caattataaa tacccaaagg aagactctgt
     6781 ccaccaccat agccatgtat ttccagtcgt cttctacctc gtttgtttca ttgtggctct
     6841 tcatgttttc tgctatgaat tgaacactct cgatgacatc ttcaacatcg gacgagtgct
     6901 ctgaattctc tgccacccac cgtgcaggct gctggcttga tcttctcttt ccaggtgctc
     6961 ctgccttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
     7021 ggaccatgtg atcgcgcttc tcgttggggt ctttgctaag cttggactgg gtgctcaggt
     7081 agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt
     7141 cggcgagctg cacgctgccg tcctcgatgt tgtggcgg
//
