LOCUS       sequence-254327-        6284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To visualize Doc2alpha D291N mutant in PC12 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6284)
  AUTHORS   Courtney NA, Briguglio JS, Bradberry MM, Greer C, Chapman ER
  TITLE     Excitatory and Inhibitory Neurons Utilize Different Ca(2+) Sensors 
            and Sources to Regulate Spontaneous Release.
  JOURNAL   Neuron. 2018 Jun 6;98(5):977-991.e5. doi: 
            10.1016/j.neuron.2018.04.022. Epub 2018 May 10.
  PUBMED    29754754
REFERENCE   2  (bases 1 to 6284)
  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"; doi: 
            "10.1016/j.neuron.2018.04.022"; journalName: "Neuron"; date: 
            "2018-06-6- 6"; volume: "98"; issue: "5"; pages: "977-991.e5"
COMMENT     Sequence Label: pEF Doc2alpha D291N mutant GFP
FEATURES             Location/Qualifiers
     source          1..6284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1285..1287,1288..1290,1291..2004)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1285..1287,1288..1290,1291..2001)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1330..1351)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1591..1610)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1938..1959
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2088..2107)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2153..2208
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2207..2226)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2569..2585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2569..2585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2582..2604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2593..2609
                     /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(2617..2623,2624..2641,2642..2647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2662..2683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2742..2937
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2788..2923
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2943..3077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2980..2999)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3034..3053
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3145..3162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3316..3904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3396..3415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4075..4866,4867..4935))
                     /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     4698..4717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4936..5040)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5102..6284
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5333..6275
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6232..6252
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 ggaattctgc agtcgacggt accgcgggcc cggatgagag gacgaagagg tgatcgaatg
       61 accatcaaca tccaggagca catggcaatc aacgtgtgcc ctggacccat caggcccatc
      121 cgccagatct ccgactactt ccctcgcaga ggaccaggac cagagggtgg cggcggagga
      181 aggggcttcg gggaagctcc agctcatctg gcccctctgg ctctggctcc acctgcagct
      241 ctccttggag ccactacacc cgacgatgga gctgaggtag acagctacga ctcggatgat
      301 accactgccc tgggcacgct ggaatttgac cttctctatg atcaggcttc ctgcatgctg
      361 cactgtagaa tcctcagggc caagggcctc aagcccatgg atttcaacgg cctggctgac
      421 ccctatgtaa agctgcacct cctgccagga gcctgcaagg ccaataagct aaaaaccaag
      481 acacagagga acacactgaa cccagtgtgg aacgaggagc tgacgtacag tggaatcaca
      541 gatgacgaca tcacccacaa ggtgctcagg atctcggtct gtgatgagga caagctgagt
      601 cacaatgaat ttattgggga gatccgagtg cccctccgcc gcctcaagcc ttcacagaag
      661 aagcatttta acatctgcct tgagcgtcag gtcccgcttc cttcgccctc ttcaatgtct
      721 gcggcgctga ggggcatatc ctgttacctg aaggagctag agcaggcaga gcagggacct
      781 gggctgttgg aagagcgcgg gcgcatcctg ctgagcctca gctacagctc tcggcggcat
      841 gggctgctgg tgggcattgt tcgctgtgct cacctggctg caatggatgt taacggctac
      901 tctaaccctt atgtgaagac gtacttgaga ccagacgtgg ataagaaatc caagcataag
      961 acgtgtgtaa agaagaagac gctgaacccg gaatttaatg aggaattctt ttatgagatg
     1021 gaactctcca ctctggctac caagaccctg gaagtcacag tctgggacta cgacattggc
     1081 aaatccaatg acttcatagg tggtgtgtct ctggggccag gagcccgggg agaggcccag
     1141 aaacactgga gagactgtct acatcagccg gacacagcag tggagcgctg gcataccctg
     1201 accagcgagc tgcccccagc agcaggggcg ttgccgttgg ccggatccgg aagttcgggg
     1261 tccagcggtt cttcaggcag ttccatggtg agcaagggcg aggagctgtt caccggggtg
     1321 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1381 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1441 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1501 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1561 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1621 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1681 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1741 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1801 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1861 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1921 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1981 ggcatggacg agctgtacaa gtaaggccgc actcctcagg tgcaggctgc ctatcagaag
     2041 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     2101 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     2161 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     2221 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     2281 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     2341 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     2401 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     2461 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     2521 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     2581 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2641 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2701 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     2761 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2821 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2881 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2941 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3001 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3061 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3121 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3181 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3241 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3301 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3361 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3421 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3481 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3541 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3601 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3661 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3721 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3781 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3841 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3901 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3961 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4021 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4081 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4141 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4201 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4261 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4321 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4381 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4441 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4501 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4561 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4621 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4681 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4741 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4801 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4861 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4921 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4981 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5041 cacatttccc cgaaaagtgc cacctgggtc gacattgatt attgactaga tcatcgcgtg
     5101 aggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     5161 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     5221 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     5281 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
     5341 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     5401 tacttccacg cccctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
     5461 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
     5521 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
     5581 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
     5641 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
     5701 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
     5761 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
     5821 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
     5881 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     5941 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     6001 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     6061 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     6121 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     6181 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     6241 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga
//
