LOCUS       sequence-175008-        6629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector template for co-expression of 
            EGFP-tagged and mCherry-tagged proteins using T2A. NLS and PTS1 can 
            be replaced by proteins of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6629)
  AUTHORS   Pan D, Klare K, Petrovic A, Take A, Walstein K, Singh P, Rondelet A,
            Bird AW, Musacchio A
  TITLE     CDK-regulated dimerization of M18BP1 on a Mis18 hexamer is necessary
            for CENP-A loading.
  JOURNAL   Elife. 2017 Jan 6;6. pii: e23352. doi: 10.7554/eLife.23352.
  PUBMED    28059702
REFERENCE   2  (bases 1 to 6629)
  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: "Elife."; 
            date: "2017-01-6"; volume: "6. pii: e23352. doi"; pages: " 
            10.7554/eLife.23352"
COMMENT     Sequence Label: pcDNA5-EGFP-NLS-T2A-mCherry-PTS1
FEATURES             Location/Qualifiers
     source          1..6629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             724..744
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             769..822
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             823..1530
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(968..986)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1236..1255
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1577..1594)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1583..1807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1940..1959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2091..2138
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2146..3168
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2146..3168
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3298..3419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3335..3354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3389..3408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3468..3484)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3468..3484)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3481..3503)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3492..3508
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3516..3522,3523..3540,3541..3546))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3561..3582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3699..3716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3870..4458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3950..3969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4629..5420,5421..5489))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5252..5271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5490..5594)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5669..5688)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5860..6239
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6240..6443
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6394..6414
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6445..6463
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6466..6484
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6494..6518
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga
      721 tcccctaaga agaagcgcaa ggtcctcgag agcggccgcg gaagcggaga gggcagagga
      781 agtctgctaa catgcggtga cgtcgaggag aatcctggac ctatggtgag caagggcgag
      841 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
      901 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      961 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     1021 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     1081 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1141 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     1201 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     1261 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1321 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1381 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     1441 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1501 tccaccggcg gcatggacga gctgtacaag gctagcggat acaagagtaa gctgtaataa
     1561 cccgggtaat gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1621 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1681 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1741 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     1801 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     1861 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1921 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1981 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2041 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta
     2101 ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc
     2161 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     2221 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     2281 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     2341 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     2401 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     2461 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     2521 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     2581 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     2641 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     2701 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     2761 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     2821 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     2881 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     2941 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     3001 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     3061 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     3121 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga
     3181 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3241 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3301 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3361 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3421 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     3481 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3541 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3601 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3661 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3721 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3781 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3961 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4021 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4081 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4141 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4201 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4261 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4321 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4381 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4441 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4501 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4561 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4621 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4681 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4741 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4801 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4861 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4921 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4981 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5041 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5101 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5161 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5221 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5281 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5341 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5401 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5461 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5521 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5581 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     5641 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5701 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     5761 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     5821 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     5881 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     5941 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     6001 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     6061 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     6121 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     6181 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     6241 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     6301 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     6361 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     6421 gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata
     6481 gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
     6541 gttttgacct ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac
     6601 ttaagcttgg taccgagctc ggatctacc
//
