LOCUS       sequence-254222-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To visualize Doc2beta D303N mutant in PC12 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  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 6286)
  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 Doc2beta D303N mutant GFP
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..1193
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          242..1184
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1141..1161
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2492..2501
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2498..2500,2501..2503,2504..3217)
                     /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(2498..2500,2501..2503,2504..3214)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2543..2564)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2804..2823)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3151..3172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3304..3323)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3369..3424
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3423..3442)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3785..3801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3785..3801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3798..3820)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3809..3825
                     /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(3833..3839,3840..3857,3858..3863))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3878..3899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      3984..4118
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4138..4272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4175..4194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4229..4248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4340..4357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4511..5099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4591..4610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5270..6061,6062..6130))
                     /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     5893..5912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6131..6235)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
       61 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
      121 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      181 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      241 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt
      301 gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc gagcttcggg
      361 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
      421 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
      481 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
      541 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
      601 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
      661 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
      721 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
      781 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
      841 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
      901 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
      961 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     1021 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     1081 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     1141 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaggaattc
     1201 caccatgacc ctccgaagac gtggagagaa ggctacaatc agcatccaag agcatatggc
     1261 aatcgacgtg tgtcctggac ctatccgacc tatcaagcag atctccgatt acttcccacg
     1321 attccctcgt ggactcccac caacagctgc accacgtgct tcagcacctc cagacgctcc
     1381 tgcacgatcg ccagcagcta ccgcaggtcc tcgtagcccc tccgacggag cacgcgacga
     1441 cgacgaagat gtggaccagc tcttcggagc ctacggtgcc agcccaggcc ccagccccgg
     1501 acccagcccc gtgaggccgc cggccaagcc ccccgaggac gaaccggacg ccgacggcta
     1561 cgagtcagac gactgcaccg ccctgggtac actggacttc agtctgctct atgaccagga
     1621 gaacaacgca ctgcactgca ccatcagcaa agccaagggc ctgaagccga tggatcacaa
     1681 tggactggct gatccctacg tcaaactaca cttgctgcct ggagccagca aggcaaataa
     1741 gctcagaaca aaaaccctac ggaacactct gaacccctcc tggaacgaga ccctcaccta
     1801 ttacgggatc acggatgagg acatgatccg aaagaccctg aggatctccg tgtgtgacga
     1861 ggacaaattc cgccacaacg agttcatcgg agagactcga gtgcccctga agaagctgaa
     1921 acccaaccac accaagacat tcagcatctg cctggagaag cagctgccgg tggacaaggc
     1981 agaggacaag tccctggagg agcgtggccg catcctcatc tctctcaagt acagctcaca
     2041 gaagcagggc ctgctggtgg gcatcgttcg ctgcgcacac ctggcggcca tggatgctaa
     2101 cggctactca aacccctacg tgaaaacata tctgaaacca gatgtagaca agaaatccaa
     2161 gcataagacc gcagtcaaga agaaaacact aaaccctgaa ttcaatgagg agttctgtta
     2221 cgagatcaag catggagacc tagccaaaaa gaccctggag gtcactgtct gggactatga
     2281 tattggaaaa tccaatgatt tcatcggtgg ggtggttctg ggcatcaatg ccaagggtga
     2341 gcgcctgaag cactggtttg actgtctgaa gaacaaggac aagaggattg agcgttggca
     2401 cacgctcacc aatgagatcc caggggctgt actcagcgac ggaagttcgg ggtccagcgg
     2461 ttcttcaggc agttcccggg atccaccggt cgccaccatg gtgagcaagg gcgaggagct
     2521 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2581 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2641 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2701 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2761 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2821 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2881 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2941 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     3001 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3061 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     3121 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3181 cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcactc ctcaggtgca
     3241 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     3301 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3361 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     3421 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     3481 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     3541 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     3601 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     3661 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     3721 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
     3781 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3841 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3901 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
     3961 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccagttccg cccattctcc
     4021 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4081 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac
     4141 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4201 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4261 catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4321 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4381 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4441 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4501 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4561 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4621 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4681 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4741 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4801 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4861 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4921 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4981 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5041 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5101 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5161 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5221 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5281 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5341 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5401 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5461 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5521 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5581 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5641 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5701 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5761 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5821 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5881 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5941 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6001 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6061 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6121 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6181 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6241 ttccccgaaa agtgccacct gggtcgacat tgattattga ctagat
//
