LOCUS       sequence-175012-        6695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High-efficient mammalian expression vector for co-expression of 
            EGFP-tagged and mCherry-tagged proteins using P2AT2A. 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 6695)
  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 6695)
  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-P2AT2A-mCherry-PTS1
FEATURES             Location/Qualifiers
     source          1..6695
                     /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             835..888
                     /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             889..1596
                     /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(1034..1052)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1302..1321
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1643..1660)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1649..1873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2006..2025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2157..2204
                     /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             2212..3234
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2212..3234
                     /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    3364..3485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3401..3420)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3455..3474
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3534..3550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3534..3550)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3547..3569)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3558..3574
                     /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(3582..3588,3589..3606,3607..3612))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3627..3648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3765..3782)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3936..4524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4016..4035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4695..5486,5487..5555))
                     /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     5318..5337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5556..5660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5735..5754)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5926..6305
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6306..6509
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6460..6480
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6511..6529
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6532..6550
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6560..6584
                     /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 gacctggaag cggagagggc
      841 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggacctat ggtgagcaag
      901 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
      961 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1021 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1081 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1141 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1201 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     1261 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     1321 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     1381 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     1441 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     1501 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     1561 cgccactcca ccggcggcat ggacgagctg tacaaggcta gcggatacaa gagtaagctg
     1621 taataacccg ggtaatgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1681 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1741 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1801 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1861 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     1921 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1981 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2041 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2101 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag
     2161 ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct
     2221 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     2281 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     2341 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     2401 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     2461 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     2521 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     2581 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     2641 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     2701 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     2761 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     2821 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     2881 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     2941 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     3001 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     3061 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     3121 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     3181 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta
     3241 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     3301 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     3361 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3421 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3481 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     3541 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3601 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3721 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3781 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3841 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3901 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3961 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4021 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4081 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4141 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4201 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4261 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4321 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4381 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4441 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4501 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4561 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4621 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4681 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4741 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4801 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4861 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4921 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4981 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5041 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5101 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5161 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5221 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5281 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5341 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5401 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5461 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5521 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5581 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5641 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     5701 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5761 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     5821 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     5881 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     5941 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     6001 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     6061 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     6121 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     6181 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     6241 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     6301 ccatggtgat gctgttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     6361 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     6421 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     6481 tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca
     6541 gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
     6601 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt
     6661 ttaaacttaa gcttggtacc gagctcggat ctacc
//
