LOCUS       sequence-69485-e        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   Richards CI, Srinivasan R, Xiao C, Mackey ED, Miwa JM, Lester HA
  TITLE     Trafficking of {alpha}4* Nicotinic Receptors Revealed by 
            Superecliptic Phluorin: EFFECTS OF A {beta}4 AMYOTROPHIC LATERAL 
            SCLEROSIS-ASSOCIATED MUTATION AND CHRONIC EXPOSURE TO NICOTINE.
  JOURNAL   J Biol Chem. 2011 Sep 9;286(36):31241-9. Epub 2011 Jul 18.
  PUBMED    21768117
REFERENCE   2  (bases 1 to 6513)
  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: "J Biol 
            Chem."; date: "2011-09-9"; volume: "286(36)"; pages: "31241-9. Epub 
            2011 Jul 18"
COMMENT     Sequence Label: pCI-neo-alpha4-SEP
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2990..3709
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MISKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3023..3051)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3646..3666
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    3857..3978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3868..3887)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3922..3941
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4164..4619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4251..4270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4461..4482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4736..5093
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4736..4927
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4759..4779)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4944..5079
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5144..5938
                     /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(5198..5217)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5808..5827
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6002..6050
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6091..6110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6210..6232
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6270..6288)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6356..6460
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc ctcgagaatt cggcttccat ggagatcggg ggctccgggg cgccgccgcc
     1141 gctgctgctc ctgccgctcc tgctgctctt agggaccggc ctcttgcctg ctagcagcca
     1201 catagagacc cgggcccatg cggaggagcg gctcctgaag agactcttct ctggctacaa
     1261 caagtggtct cggccagtag ccaatatctc agatgtggtc cttgtccgct ttggcttgtc
     1321 gattgctcag ctcattgatg tggatgagaa aaaccagatg atgacgacca acgtgtgggt
     1381 gaagcaggag tggcatgact acaaactgcg ctgggaccct ggtgactacg agaatgtcac
     1441 ctccatccgc atcccatctg aactcatctg gaggcctgac atcgtcctct acaacaacgc
     1501 ggacggggac tttgcagtca cccacctaac caaagcccac ctgttctatg atgggcgtgt
     1561 acagtggaca cccccggcca tctataagag ctcctgcagc atcgacgtca ccttcttccc
     1621 cttcgaccag cagaactgta ccatgaagtt tgggtcctgg acctacgaca aggccaagat
     1681 tgacttggtg agcatgcaca gccgtgtgga ccaactggac ttctgggaaa gtggggagtg
     1741 ggtcattgtg gatgccgtgg gcacctacaa caccaggaag tatgaatgct gtgccgagat
     1801 ctatcctgac atcacctacg ccttcatcat ccgccgactg ccactgttct acaccatcaa
     1861 ccttatcatc ccgtgcctgc tcatctcctg cctcaccgtg ctggtcttct atctgccctc
     1921 ggagtgcggc gagaaggtca cgctgtgcat ctcggtgctg ctttctctca ccgtcttcct
     1981 gctgctcatc accgaaatca tcccgtccac ctcgctggtc atcccgctca tcggcgagta
     2041 cctgctcttc accatgatct tcgtcaccct ctccattgtc atcacggtct tcgtgctcaa
     2101 tgtacaccac cgctcaccac gcacacacac catgcccgcc tgggtgcgca gagtcttcct
     2161 ggacattgtg ccccgtctcc tcttcatgaa gcgcccatct gtggtcaaag acaactgccg
     2221 gagacttatc gaatccatgc acaagatggc caacgcccct cgtttctggc cagagcctga
     2281 gagtgagccc ggcatcttgg gtgacatctg caaccaaggc ctgtcacctg ccccaacttt
     2341 ctgcaaccgc atggacacag cagtcgagac ccagcctaca tgcaggtcac cctcccacaa
     2401 ggtccctgac ttgaagacat cagaggttga gaaggccagt ccctgtccat cacctggctc
     2461 ttgtcaccca cccaatagca gtggggcccc agtgctcatc aaagccaggt ccctgagcgt
     2521 ccagcatgtg cccagctccc aggaagcagc cgagggcagc atccgctgcc ggtctcggag
     2581 tatccagtac tgtgtttccc aagatggagc tgcttccctg actgagagca agcccactgg
     2641 ctccccagcc tccctgaaga cccgtccatc ccagcttcca gtgtcagacc agacctctcc
     2701 atgcaaatgc acatgcaagg aaccatctcc tgtgtccccc atcactgtgc tcaaggctgg
     2761 aggcaccaaa gcacctcccc aacacctgcc cctgtcacca gccctgacac gggcagtaga
     2821 aggcgtccag tacattgcag accacctcaa ggcagaagac acagacttct cggtgaagga
     2881 ggactggaaa tacgtggcca tggtcattga ccgaatcttc ctctggatgt tcatcattgt
     2941 ctgccttctg ggcactgtgg gactcttcct gcctccatgg ttggctggta tgatcagtaa
     3001 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     3061 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
     3121 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
     3181 tttaacttat ggtgttcaat gcttttcaag atacccagat catatgaaac ggcatgactt
     3241 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
     3301 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
     3361 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
     3421 caactataac gatcaccagg tgtacatcat ggcagacaaa caaaagaatg gaatcaaagc
     3481 taacttcaaa attagacaca acattgaaga tggaggcgtt caactagcag accattatca
     3541 acaaaatact ccaattggcg atgggcccgt ccttttacca gacaaccatt acctgtttac
     3601 aacttctact ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     3661 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataag gaatagcggc
     3721 acctagctcc caggtctcta cagggccatg cgactcgtca gtccacccac aagggcgaat
     3781 tcacgcgtgg tacctctaga gtcgacccgg gcggccgctt ccctttagtg agggttaatg
     3841 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     3901 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     3961 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     4021 gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatccgataa
     4081 ggatcgatcc gggctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4141 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4201 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4261 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4321 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4381 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4441 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4501 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4561 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     4621 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgcggatct
     4681 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag
     4741 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4801 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4861 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4921 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4981 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5041 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg
     5101 attcttctga cacaacagtc tcgaacttaa ggctagagcc accatgattg aacaagatgg
     5161 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5221 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5281 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     5341 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5401 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5461 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5521 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5581 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5641 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     5701 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5761 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5821 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5881 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5941 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgatggccg
     6001 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagc
     6061 gataaggatc cgcgtatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6121 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     6181 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     6241 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     6301 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     6361 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6421 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6481 tgtcgccctt attccctttt ttgcggcatt ttg
//
