LOCUS       sequence-391804-        6973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes N-terminal eGFP-tagged gephyrin including the C4a splice 
            cassette for expression in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6973)
  AUTHORS   Campbell BFN, Dittmann A, Dreier B, Pluckthun A, Tyagarajan SK
  TITLE     A DARPin-based molecular toolset to probe gephyrin and inhibitory 
            synapse biology.
  JOURNAL   Elife. 2022 Oct 31;11:e80895. doi: 10.7554/eLife.80895.
  PUBMED    36314779
REFERENCE   2  (bases 1 to 6973)
  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: "Elife."; 
            date: "2022-10-31"; volume: "11:e80895. doi"; pages: " 
            10.7554/eLife.80895"
COMMENT     Sequence Label: pEGFPC2-gephyrin C4a
FEATURES             Location/Qualifiers
     source          1..6973
                     /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    3761..3882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3798..3817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3852..3871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3889..4344)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4026..4047)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4238..4257
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4371..4475
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4477..4834
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4477..4668
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4500..4520)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4685..4820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4869..5663
                     /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             4869..5663
                     /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(4923..4942)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5533..5552
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5851..5870)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5895..5942
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6271..6859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6760..6779
                     /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 agctcaatgg cgaccgaggg aatgatcctc
     1381 acaaaccacg accatcaaat ccgtgtcgga gtcctcacag tgagtgatag ctgcttcagg
     1441 aatcttgcag aagaccgcag tgggataaat ctaaaagatc tcgtccagga tccttctttg
     1501 ttgggtggga ctatatcagc atacaagata gtaccagatg aaatagaaga aatcaaggaa
     1561 acactcatag actggtgtga tgagaaggaa cttaatttga tattaacaac tggcggaaca
     1621 gggtttgcac cacgagatgt cactccagag gccacaaaag aagtaataga acgggaagcg
     1681 ccagggatgg ccctggcaat gctgatggga tcacttaatg ttgcacctct gggcatgctc
     1741 tctagaccag tgtgtggaat aagagggaaa actctgataa ttaacctgcc tggtagcaag
     1801 aaaggatctc aggaatgctt tcagttcata ctgccagctc tacctcatgc cattgacctt
     1861 ttacgtgatg ccattgtaaa agtaaaggag gtgcatgatg aacttgaaga tttaccttcc
     1921 ccaccacctc ctctttctcc acctcctaca actagcccac ataagcagac agaagacaaa
     1981 ggagttcagt gtgaagagga ggaagaagaa aagaaagaca gtggtgtagc ttcgacagaa
     2041 gatagttcct catcacatat tactgcagca gctcttgctg caaagattcc agactccatc
     2101 atttctcgtg gcgttcaggt gctcccacga gacacagcct cccttagcac tactccttca
     2161 gagtcgcccc gtgcccaggc tacatctcgc ctctctacag cttcttgtcc aacaccaaaa
     2221 caaattagac ggccggatga aagcaaagga gttgctagta gagttggatc cctcaaagtc
     2281 cagtccaggt gcagcagcaa ggagaacatt ctaagagcca gtcacagtgc tgtagatatc
     2341 accaaggtgg ctagaagaca tcgcatgtct ccttttcctc tgacgtctat ggacaaagcc
     2401 ttcattacag ttctggagat gactccggtg cttggtacag aaatcatcaa ttaccgagat
     2461 ggaatggggc gagtccttgc tcaagatgtg tatgcaaaag acaacctacc cccattccca
     2521 gcatcagtaa aagatggcta tgctgttcga gctgctgatg gtccaggaga tcgtttcatc
     2581 attggggaat cccaagctgg tgaacagcca actcagacag taatgcccgg acaagtgatg
     2641 cgggttacaa caggtgctcc aatcccctgc ggtgctgatg cagtagtgca agttgaagat
     2701 actgaactca tcagggaatc agatgatggt actgaagaac ttgaagtgcg aattctggtg
     2761 caagctcggc caggccaaga catcagaccc atcggccacg acattaaaag aggggagtgt
     2821 gttttggcaa aaggaaccca tatgggccct tcagaaattg gtctccttgc aactgtaggt
     2881 gtcacagaag ttgaagttaa taagtttcca gtggttgccg ttatgtcaac agggaatgag
     2941 ctactaaatc ctgaagatga cctcttacca ggaaagattc gggacagcaa tcgatcaaca
     3001 cttctagcaa caattcagga acatggttac ccaacaatca acttgggaat tgtaggagac
     3061 aacccagatg acttactcaa tgccttgaat gagggtatca gccgtgctga tgtcatcatc
     3121 acgtcaggag gtgtatccat gggggaaaag gactatctca agcaggtgct ggacatcgat
     3181 cttcatgctc agatccattt tggcagggtt tttatgaagc caggcttgcc aacaacattt
     3241 gcaactttgg atattgatgg tgtaagaaaa attatttttg cactaccagg gaatcctgta
     3301 tcagctgtgg tcacctgcaa cctctttgtt gtacccgcac tgagaaaaat gcagggtatc
     3361 ttggatcctc ggccaaccat catcaaagct aggttatcat gtgacgtaaa gctggaccct
     3421 cgcccagaat accaccggtg tatactgact tggcatcacc aagaaccact gccctgggcc
     3481 cagagtacag gtaatcagat gagcagccgt ctgatgagca tgcgcagtgc caatggattg
     3541 ctgatgctac ctccaaagac agagcagtat gtggaacttc acaaaggcga ggtggtggac
     3601 gtcatggtca tcggacggta ccgcgggccc gggatccacc ggatctagat aactgatcat
     3661 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3721 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3781 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3841 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg
     3901 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3961 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4021 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4081 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4141 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4201 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4261 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4321 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4381 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4441 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     4501 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4561 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     4621 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4681 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4741 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4801 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     4861 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4921 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4981 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5041 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5101 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5161 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5221 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5281 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5341 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     5401 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5461 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5521 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5581 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5641 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5701 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5761 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5821 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     5881 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     5941 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     6001 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     6061 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     6121 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6181 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6901 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6961 taccgccatg cat
//
