LOCUS       sequence-175010-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector template for co-expression of 
            EGFP-tagged and mCherry-tagged proteins using P2A. 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 6632)
  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 6632)
  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-P2A-mCherry-PTS1
FEATURES             Location/Qualifiers
     source          1..6632
                     /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..825
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             826..1533
                     /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(971..989)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1239..1258
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1580..1597)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1586..1810
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1943..1962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2094..2141
                     /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             2149..3171
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2149..3171
                     /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    3301..3422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3338..3357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3392..3411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3471..3487)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3471..3487)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3484..3506)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3495..3511
                     /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(3519..3525,3526..3543,3544..3549))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3564..3585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3702..3719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3873..4461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3953..3972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4632..5423,5424..5492))
                     /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     5255..5274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5493..5597)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5672..5691)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5863..6242
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6243..6446
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6397..6417
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6448..6466
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6469..6487
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6497..6521
                     /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 gaagcggagc tactaacttc
      781 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggt gagcaagggc
      841 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
      901 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      961 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1021 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1081 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1141 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1201 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     1261 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1321 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1381 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1441 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1501 cactccaccg gcggcatgga cgagctgtac aaggctagcg gatacaagag taagctgtaa
     1561 taacccgggt aatgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1621 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1681 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1741 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1801 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     1861 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1921 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1981 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2041 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     2101 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     2161 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     2221 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     2281 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     2341 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     2401 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     2461 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     2521 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     2581 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     2641 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     2701 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     2761 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     2821 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     2881 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     2941 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     3001 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     3061 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     3121 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     3181 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3241 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3301 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3361 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3421 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     3481 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3541 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3601 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3661 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3721 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3781 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3841 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3901 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3961 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4021 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4081 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4141 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4201 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4261 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4321 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4381 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4441 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4501 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4561 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4621 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4681 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4741 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4801 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4861 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4921 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4981 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5041 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5101 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5161 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5221 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5281 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5341 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5401 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5461 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5521 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5581 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     5641 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5701 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     5761 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     5821 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     5881 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     5941 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     6001 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     6061 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     6121 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     6181 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6241 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     6301 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     6361 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     6421 ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg
     6481 atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     6541 gctgttttga cctccataga agacaccggg accgatccag cctccggact ctagcgttta
     6601 aacttaagct tggtaccgag ctcggatcta cc
//
