LOCUS       sequence-462327-        6150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of Connexin 32 and EGFP from a bicistronic 
            mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  AUTHORS   Carrer A, Leparulo A, Crispino G, Ciubotaru CD, Marin O, Zonta F, 
            Bortolozzi M
  TITLE     Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X
            mutation that causes Charcot-Marie-Tooth disease.
  JOURNAL   Hum Mol Genet. 2017 Oct 25. pii: 4565496. doi: 10.1093/hmg/ddx386.
  PUBMED    29077882
REFERENCE   2  (bases 1 to 6150)
  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: "Hum Mol 
            Genet. 2017 Oct 25. pii: 4565496. doi: 10.1093/hmg/ddx386."
COMMENT     Sequence Label: pIRES2Cx32WTEGFP
FEATURES             Location/Qualifiers
     source          1..6150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(1449..2023,2024..2026,2027..2035)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1616..1633)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1843..1862
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2036..2038,2039..2041,2042..2755)
                     /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(2036..2038,2039..2041,2042..2752)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2081..2102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2342..2361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2689..2710
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2878..2999
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2915..2934)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2969..2988
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3006..3461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3143..3164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3355..3374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3488..3592
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3594..3951
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3594..3785
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3617..3637)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3802..3937
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3986..4780
                     /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             3986..4780
                     /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(4040..4059)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4650..4669
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4968..4987)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5012..5059
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5388..5976
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5877..5896
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggactcag atctcgagat gaactggaca ggtttgtaca ccttgctcag tggcgtgaac
      601 cggcattcta ctgccattgg ccgagtatgg ctctcggtca tcttcatctt cagaatcatg
      661 gtgctggtgg tggctgcaga gagtgtgtgg ggtgatgaga aatcttcctt catctgcaac
      721 acactccagc ctggctgcaa cagcgtttgc tatgaccaat tcttccccat ctcccatgtg
      781 cggctgtggt ccctgcagct catcctagtt tccaccccag ctctcctcgt ggccatgcac
      841 gtggctcacc agcaacacat agagaagaaa atgctacggc ttgagggcca tggggacccc
      901 ctacacctgg aggaggtgaa gaggcacaag gtccacatct cagggacact gtggtggacc
      961 tatgtcatca gcgtggtgtt ccggctgttg tttgaggccg tcttcatgta tgtcttttat
     1021 ctgctctacc ctggctatgc catggtgcgg ctggtcaagt gcgacgtcta cccctgcccc
     1081 aacacagtgg actgcttcgt gtcccgcccc accgagaaaa ccgtcttcac cgtcttcatg
     1141 ctagctgcct ctggcatctg catcatcctc aatgtggccg aggtggtgta cctcatcatc
     1201 cgggcctgtg cccgccgagc ccagcgccgc tccaatccac cttcccgcaa gggctcgggc
     1261 ttcggccacc gcctctcacc tgaatacaag cagaatgaga tcaacaagct gctgagtgag
     1321 caggatggct ccctgaaaga catactgcgc cgcagccctg gcaccggggc tgggctggct
     1381 gaaaagagcg accgctgctc ggcctgctga gaattctgca gtcgacggta ccgcgggccc
     1441 gggatccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     1501 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     1561 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     1621 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     1681 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     1741 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     1801 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     1861 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     1921 cctcggtaca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     1981 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatggt
     2041 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2101 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2161 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2221 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2281 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2341 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2401 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2461 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2521 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2581 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2641 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2701 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     2761 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2821 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2881 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2941 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     3001 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3061 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     3481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3541 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     3601 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3661 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3721 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3781 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3841 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3901 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3961 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4021 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4081 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4141 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4201 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4261 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4321 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4381 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4441 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4501 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4561 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4621 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4681 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4741 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4801 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4861 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4921 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4981 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     5041 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     5101 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     5161 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     5221 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5281 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5341 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5401 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5461 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5521 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5581 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5641 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5701 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5761 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5821 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5881 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5941 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6001 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6061 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
     6121 attagttcat agcccatata tggagttccg
//
