LOCUS       sequence-127476-        5525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human Cx30 (Gjb6 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 5525)
  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 5525)
  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-Cx30
FEATURES             Location/Qualifiers
     source          1..5525
                     /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    2313..2434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2350..2369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2404..2423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2441..2896)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2578..2599)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2790..2809
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2923..3027
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3029..3386
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3029..3220
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3052..3072)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3237..3372
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3421..4215
                     /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             3421..4215
                     /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(3475..3494)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4085..4104
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4403..4422)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4447..4494
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4823..5411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5312..5331
                     /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 gggggacgct gcacactttc
     1381 atcgggggtg tcaacaaaca ctccaccagc atcgggaagg tgtggatcac agtcatcttt
     1441 attttccgag tcatgatcct cgtggtggct gcccaggaag tgtggggtga cgagcaagag
     1501 gacttcgtct gcaacacact gcaaccggga tgcaaaaatg tgtgctatga ccactttttc
     1561 ccggtgtccc acatccggct gtgggccctc cagctgatct tcgtctccac cccagcgctg
     1621 ctggtggcca tgcatgtggc ctactacagg cacgaaacca ctcgcaagtt caggcgagga
     1681 gagaagagga atgatttcaa agacatagag gacattaaaa agcagaaggt tcggatagag
     1741 gggtcgctgt ggtggacgta caccagcagc atctttttcc gaatcatctt tgaagcagcc
     1801 tttatgtatg tgttttactt cctttacaat gggtaccacc tgccctgggt gttgaaatgt
     1861 gggattgacc cctgccccaa ccttgttgac tgctttattt ctaggccaac agagaagacc
     1921 gtgtttacca tttttatgat ttctgcgtct gtgatttgca tgctgcttaa cgtggcagag
     1981 ttgtgctacc tgctgctgaa agtgtgtttt aggagatcaa agagagcaca gacgcaaaaa
     2041 aatcacccca atcatgccct aaaggagagt aagcagaatg aaatgaatga gctgatttca
     2101 gatagtggtc aaaatgcaat cacaggtttc ccaagctaag agctcaagct tcgaattctg
     2161 cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc
     2221 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2281 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2341 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2401 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2461 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2521 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2581 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2641 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2701 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2761 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2821 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2881 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2941 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3001 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3061 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3121 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3181 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3241 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3301 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3361 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3421 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3481 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3541 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3601 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3661 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3721 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3781 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3841 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3901 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3961 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4021 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4081 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4141 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4201 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4261 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4321 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4381 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4441 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4501 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4561 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4621 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4681 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4741 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4801 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4861 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4921 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4981 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5041 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5101 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5161 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5221 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5281 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5341 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5401 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5461 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     5521 tgcat
//
