LOCUS       sequence-350245-        5129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-express in mammalian cells two target proteins, using an internal
            ribosome entry site and two multi-cloning sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5129)
  AUTHORS   Carman PJ, Barrie KR, Dominguez R
  TITLE     Novel human cell expression method reveals the role and prevalence 
            of posttranslational modification in nonmuscle tropomyosins.
  JOURNAL   J Biol Chem. 2021 Oct;297(4):101154. doi: 10.1016/j.jbc.2021.101154.
            Epub 2021 Sep 1.
  PUBMED    34478714
REFERENCE   2  (bases 1 to 5129)
  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."; date: "2021-10"; volume: "297(4):101154. doi"; pages: " 
            10.1016/j.jbc.2021.101154. Epub 2021 Sep 1"
COMMENT     Sequence Label: pJC7
FEATURES             Location/Qualifiers
     source          1..5129
                     /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"
     misc_feature    join(683..1256,1257..1259,1260..1268)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(850..867)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1077..1096
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     misc_feature    1312..1900
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1365..1385)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1783..1794)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1917..2038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1954..1973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2008..2027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2045..2500)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2182..2203)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2394..2413
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2527..2631
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2633..2990
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2633..2824
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2656..2676)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2841..2976
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3025..3819
                     /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             3025..3819
                     /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(3079..3098)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3689..3708
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4007..4026)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4051..4098
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4427..5015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4916..4935
                     /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 gctagctcgc
      601 gagtcgacgg cggccgcgaa ttcctcgagt cttgatcact aactagtaat accggtactc
      661 tgcaggaagg ggatccaatt cgcccctctc cctccccccc ccctaacgtt actggccgaa
      721 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
      781 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
      841 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
      901 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
      961 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     1021 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     1081 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     1141 gatctgatct ggggcctcgg tgcacatgct ttacgtgtgt ttagtcgagg ttaaaaaacg
     1201 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     1261 ccacaacctc cggatatccc gggatcagat ctatcgagct catttctaga taatcaacct
     1321 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     1381 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     1441 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     1501 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     1561 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     1621 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     1681 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     1741 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     1801 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     1861 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcaattgtt gttgttaact
     1921 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     1981 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac
     2041 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2101 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2161 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2221 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2281 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2341 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2401 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2461 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     2521 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2581 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     2641 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2701 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2761 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2821 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2881 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     2941 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     3001 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3061 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3121 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3181 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     3241 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3301 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3361 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3421 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3481 gtcgatcagg atgatctgga cgaggagcat caggggctcg cgccagccga actgttcgcc
     3541 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3601 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3661 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaggagctt
     3721 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3781 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3841 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3901 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3961 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     4021 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     4081 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     4141 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     4201 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     4261 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4321 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4381 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4441 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4501 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4561 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4621 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4681 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4741 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4801 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4861 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4921 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4981 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5041 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5101 ctgtggataa ccgtattacc gccatgcat
//
