LOCUS       sequence-127473-        5420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rat Cx26 (Gjb2 CDS) with an 8 amino acid linker on the 
            N-terminus of the Connexin linking to Enhanced Blue Fluorescent 
            Protein 2 (FP fused to NT of connexin). CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5420)
  AUTHORS   Stout RF Jr, Snapp EL, Spray DC
  TITLE     Connexin Type and Fluorescent Protein-fusion Tag Determine 
            Structural Stability of Gap Junction Plaques.
  JOURNAL   J Biol Chem. 2015 Aug 11. pii: jbc.M115.659979.
  PUBMED    26265468
REFERENCE   2  (bases 1 to 5420)
  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. 2015 Aug 11. pii: jbc.M115.659979."
COMMENT     Sequence Label: EBFP2-Cx26
FEATURES             Location/Qualifiers
     source          1..5420
                     /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 blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    2208..2329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2245..2264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2299..2318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2336..2791)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2473..2494)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2685..2704
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2818..2922
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2924..3281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2924..3115
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2947..2967)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3132..3267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3316..4110
                     /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             3316..4110
                     /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(3370..3389)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3980..3999
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4298..4317)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4342..4389
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4718..5306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5207..5226
                     /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 tgaggggcga gggcgagggc
      721 gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgagc cacggcgtgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcgtcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaacttc aacagccaca acatctatat catggccgtc
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtgga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacagcc actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttccgcacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct cagatggatt ggggcacact acagagcatc
     1381 ctcgggggtg tcaacaagca ctccaccagc attgggaaaa tctggctcac tgtcctcttc
     1441 atcttccgca tcatgatcct cgtggtggcc gcgaaggagg tgtggggaga tgagcaagcc
     1501 gattttgttt gcaacactct ccagcctggc tgtaagaatg tgtgctacga ccactacttc
     1561 cccatctctc acatccggct ctgggctctg cagctgatca tggtgtccac gccggccctc
     1621 ctggtagcta tgcacgtggc ctaccggaga cacgaaaaga aacggaagtt catgaaggga
     1681 gagataaaga acgagtttaa ggacatcgaa gagatcaaaa cccagaaggt ccgtatcgaa
     1741 gggtccctgt ggtggaccta caccaccagc atcttcttcc gggtcatctt cgaagctgtc
     1801 ttcatgtatg tcttttacat catgtacaat ggcttcttca tgcagcgtct ggtgaagtgt
     1861 aacgcctggc cttgtcccaa tacagtggac tgcttcattt ccaggcccac agaaaagact
     1921 gtcttcacgg tgttcatgat ctctgtgtct ggaatttgca tcctgctaaa catcacagag
     1981 ctgtgctatc tgttcattag gtattgctca gggaagtcca aaagaccagt ctaagagctc
     2041 aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccaccgga tctagataac
     2101 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2161 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2221 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2281 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     2341 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2401 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2461 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2521 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2581 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2641 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2701 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2761 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2821 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2881 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2941 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3001 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3061 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3121 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3181 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3241 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     3301 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3361 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3421 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3481 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3541 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3601 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3661 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3721 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3781 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3841 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3901 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3961 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4021 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4081 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4141 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4201 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4261 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     4321 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     4381 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     4441 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     4501 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     4561 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4861 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5401 accgtattac cgccatgcat
//
