LOCUS       sequence-391785-        6942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes N-terminal eGFP-tagged P1-gephyrin S268/270E for expression 
            in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6942)
  AUTHORS   Tyagarajan SK, Ghosh H, Yevenes GE, Imanishi SY, Zeilhofer HU, 
            Gerrits B, Fritschy JM
  TITLE     Extracellular signal-regulated kinase and glycogen synthase kinase 
            3beta regulate gephyrin postsynaptic aggregation and GABAergic 
            synaptic function in a calpain-dependent mechanism.
  JOURNAL   J Biol Chem. 2013 Apr 5;288(14):9634-47. doi: 
            10.1074/jbc.M112.442616. Epub 2013 Feb 13.
  PUBMED    23408424
REFERENCE   2  (bases 1 to 6942)
  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: 
            "10.1074/jbc.M112.442616"; journalName: "J Biol Chem"; date: 
            "2013-04-5- 5"; volume: "288"; issue: "14"; pages: "9634-47"
COMMENT     Sequence Label: pEGFPC2-gephyrin S268E/270E
FEATURES             Location/Qualifiers
     source          1..6942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3730..3851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3767..3786)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3821..3840
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3858..4313)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3995..4016)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4207..4226
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4340..4444
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4446..4803
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4446..4637
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4469..4489)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4654..4789
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4838..5632
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4838..5632
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4892..4911)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5502..5521
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5820..5839)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5864..5911
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6240..6828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6729..6748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggccggac tcagatctcg agctcaagct tcgaattcat ggcgaccgag
     1381 ggaatgatcc tcacaaacca cgaccatcaa atccgtgtcg gagtcctcac agtgagtgat
     1441 agctgcttca ggaatcttgc agaagaccgc agtgggataa atctaaaaga tctcgtccag
     1501 gatccttctt tgttgggtgg gactatatca gcatacaaga tagtaccaga tgaaatagaa
     1561 gaaatcaagg aaacactcat agactggtgt gatgagaagg aacttaattt gatattaaca
     1621 actggcggaa cagggtttgc accacgagat gtcactccag aggccacaaa agaagtaata
     1681 gaacgggaag cgccagggat ggccctggca atgctgatgg gatcacttaa tgttacacct
     1741 ctgggcatgc tctctagacc agtgtgtgga ataagaggga aaactctgat aattaacctg
     1801 cctggtagca agaaaggatc tcaggaatgc tttcagttca tactgccagc tctacctcat
     1861 gccattgacc ttttacgtga tgccattgta aaagtaaagg aggtgcatga tgaacttgaa
     1921 gatttacctt ccccaccacc tcctctttct ccacctccta caactagccc acataagcag
     1981 acagaagaca aaggagttca gtgtgaagag gaggaagaag aaaagaaaga cagtggcgta
     2041 gcttcgacag aagatagttc ctcatcacat attactgcag cagctcttgc tgcaaagatt
     2101 ccagactcca tcatttctcg tggcgttcag gtgctcccac gagacacagc ctcccttagc
     2161 actactcctg aagaggagcc ccgtgcccag gctacatctc gcctctctac agcttcttgt
     2221 ccaacaccaa aagtccagtc caggtgcagc agcaaggaga acattctaag agccagtcac
     2281 agtgctgtag atatcaccaa ggtggctaga agacatcgca tgtctccttt tcctctgacg
     2341 tctatggaca aagccttcat tacagttctg gagatgactc cggtgcttgg tacagaaatc
     2401 atcaattacc gagatggaat ggggcgagtc cttgctcaag atgtgtatgc aaaagacaac
     2461 ctacccccat tcccagcatc agtaaaagat ggctatgctg ttcgagctgc tgatggtcca
     2521 ggagatcgtt tcatcattgg ggaatcccaa gctggtgaac agccaactca gacagtaatg
     2581 cccggacaag tgatgcgggt tacaacaggt gctccaatcc cctgcggtgc tgatgcagta
     2641 gtgcaagttg aagatactga actcatcagg gaatcagatg atggtactga agaacttgaa
     2701 gtgcgcattc tggtgcaagc tcggccaggc caagacatca gacccatcgg ccacgacatt
     2761 aaaagagggg agtgtgtttt ggcaaaagga acccatatgg gcccttcaga aattggtctc
     2821 cttgcaactg taggtgtcac agaagttgaa gttaataagt ttccagtggt tgccgttatg
     2881 tcaacaggga atgagctact aaatcctgaa gatgacctct taccaggaaa gattcgggac
     2941 agcaatcgat caacacttct agcaacaatt caggaacatg gttacccaac aatcaacttg
     3001 ggaattgtag gagacaaccc agatgactta ctcaatgcct tgaatgaggg tatcagccgt
     3061 gctaatgtca tcatcacgtc aggaggtgta tccatggggg aaaaggacta tctcaagcag
     3121 gtgctggaca tcgatcttca tgctcagatc cattttggca gggtttttat gaagccaggc
     3181 ttgccaacaa catttgcaac tttggatatt gatggtgtaa gaaaaattat ttttgcacta
     3241 ccagggaatc ctgtatcagc tgtggtcacc tgcaacctct ttgttgtacc cgcactgaga
     3301 aaaatgcagg gtatcttgga tcctcggcca accatcatca aagctaggtt atcatgtgac
     3361 gtaaagctgg accctcgccc agaataccac cggtgtatac tgacttggca tcaccaagaa
     3421 ccactgccct gggcccagag tacaggtaat cagatgagca gccgtctgat gagcatgcgc
     3481 agtgccaatg gattgctgat gctacctcca aagacagagc agtatgtgga acttcacaaa
     3541 ggcgaggtgg tggacgtcat ggtcatcgga cggctatgac tcgacggtac cgcgggcccg
     3601 ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta
     3661 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3721 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3781 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3841 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3901 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3961 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4021 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4081 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4141 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4201 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4261 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     4321 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4381 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4441 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4501 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4561 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4621 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4681 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4741 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4801 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4861 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4921 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4981 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     5041 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     5101 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     5161 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     5221 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5281 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     5341 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     5401 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     5461 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     5521 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5581 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     5641 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5701 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5761 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     5821 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5881 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5941 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     6001 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     6061 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     6121 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6361 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6901 ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
