LOCUS       sequence-334720-        5159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-express in mammalian cells two target proteins C-terminally fused
            to V5 and FLAG tags respectively, 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 5159)
  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 5159)
  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: pJC8
FEATURES             Location/Qualifiers
     source          1..5159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..160
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             364..387
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    407..995
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(460..480)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(878..889)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1012..1133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1049..1068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1103..1122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1140..1595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1277..1298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1489..1508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1622..1726
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1728..2085
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1728..1919
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1751..1771)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1936..2071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2120..2914
                     /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             2120..2914
                     /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(2174..2193)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2784..2803
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3102..3121)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3146..3193
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3522..4110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4011..4030
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4285..4588
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4589..4792
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4743..4763
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4838..4879
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    join(4906..5159,1..320,321..323,324..332)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5073..5090)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
ORIGIN
        1 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
       61 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
      121 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
      181 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacgt
      241 gtgtttagtc gaggttaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
      301 ttgaaaaaca cgatgataat atggccacaa ccttctcgtc gacgcggccg cgaattcgct
      361 agcgactaca aagacgatga cgacaagtaa gagctcattt ctagataatc aacctctgga
      421 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
      481 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
      541 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
      601 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
      661 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
      721 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
      781 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
      841 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
      901 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
      961 gacgagtcgg atctcccttt gggccgcctc cccgcatcaa ttgttgttgt taacttgttt
     1021 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1081 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta
     1141 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     1201 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     1261 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1321 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1381 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1441 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1501 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1561 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     1621 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1681 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     1741 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     1801 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     1861 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     1921 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     1981 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2041 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     2101 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2161 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2221 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2281 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2341 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     2401 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     2461 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     2521 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     2581 tcaggatgat ctggacgagg agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     2641 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     2701 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     2761 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgagg agcttggcgg
     2821 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     2881 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     2941 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3001 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3061 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     3121 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     3181 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     3241 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     3301 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     3361 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3421 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3481 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3541 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3601 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3661 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3721 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3781 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3841 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3901 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3961 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4021 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4081 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4141 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4201 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     4261 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     4321 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     4381 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     4441 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     4501 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     4561 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     4621 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     4681 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     4741 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     4801 gaaccgtcag atccttctcc tcgagagatc ttccggaggt aagcctatcc ctaaccctct
     4861 cctcggtctc gattctacgt aactgcagga aggggatcca attcgcccct ctccctcccc
     4921 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat
     4981 gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
     5041 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt
     5101 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtag
//
