LOCUS       sequence-361614-        7219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for mKate2-3xNLS from the CAG promoter. 
            Confers puromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7219)
  AUTHORS   Malaguti M, Nistor PA, Blin G, Pegg A, Zhou X, Lowell S
  TITLE     Bone morphogenic protein signalling suppresses differentiation of 
            pluripotent cells by maintaining expression of E-Cadherin.
  JOURNAL   Elife. 2013 Dec 17;2:e01197. doi: 10.7554/eLife.01197.
  PUBMED    24347544
REFERENCE   2  (bases 1 to 7219)
  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: "2013-12-17"; volume: "2:e01197. doi"; pages: " 
            10.7554/eLife.01197"
COMMENT     Sequence Label: pPyCAG-mKate2-3xNLS-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    309..443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        457..836
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        838..1113
                     /label=chicken beta-actin promoter
     intron          1114..2129
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2053..2075
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2137..2156
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2193..2202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2199..2894
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             2910..2930
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2934..2954
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2958..2978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    join(3025..3599,3600..3602,3603..3611)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3192..3209)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3419..3438
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3612..4211
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3612..3631)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4108..4128
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4292..4309)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4298..4522
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4569..4585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4569..4585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4582..4604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4593..4609
                     /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(4617..4623,4624..4641,4642..4647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4662..4683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4742..4937
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4788..4923
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4943..5077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5145..5162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5316..5904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5396..5415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6075..6866,6867..6935))
                     /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     6698..6717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agctgttgtc atagtaatga ttgggtggaa acattccagg cctgggtgga gaggcttttt
       61 gcttcctctt gcaaaaccac actgacattc caggcctggg tggagaggct ttttgcttcc
      121 tcttgcaaaa ccacactgcc ctctggaggg cagttgccta gcaactaatt aaaagaggat
      181 gtcgcacggc cagctgcggt cagttagtca cttcctgctt aactgacttg acattttcta
      241 ttttaagagt cgggaggaaa attactgtgt tggaggccct ccgccatctt ctgaagctga
      301 atcgaattaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      361 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      421 atgtatctta tcatgtctgg atctgatatc atcgtcgaca ttgattattg actagttatt
      481 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      541 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      601 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      661 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      721 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      781 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
      841 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
      901 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggsgc
      961 gcgccrggsg gggsggggsg gggsgrgggg sggggsgggg cgaggcggag aggtgcggcg
     1021 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1081 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     1141 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     1201 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
     1261 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
     1321 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
     1381 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1441 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cgggggggct
     1501 gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg
     1561 gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc
     1621 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     1681 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg
     1741 gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     1801 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     1861 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     1921 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     1981 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     2041 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     2101 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     2161 ctcatcattt tggcaaagaa ttcctcgagg ccgccaccat ggtgagcgag ctgattaagg
     2221 agaacatgca catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca
     2281 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggcggtcg
     2341 agggcggccc tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca
     2401 aaaccttcat caaccacacc cagggcatcc ccgacttctt taagcagtcc ttccccgagg
     2461 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     2521 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc
     2581 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggcctcc accgagaccc
     2641 tgtaccccgc tgacggcggc ctggaaggca gagccgacat ggccctgaag ctcgtgggcg
     2701 ggggccacct gatctgcaac ttgaagacca catacagatc caagaaaccc gctaagaacc
     2761 tcaagatgcc cggcgtctac tatgtggaca gaagactgga aagaatcaag gaggccgaca
     2821 aagagaccta cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca
     2881 aactggggca cagaggcgcc acaggtagtc caaaaaagaa gagaaaggta gatccaaaaa
     2941 agaagagaaa ggtagatcca aaaaagaaga gaaaggtatt ataagcggcc gctcgataag
     3001 cttgatatcg aattccgccc cccccccctc tccctccccc ccccctaacg ttactggccg
     3061 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     3121 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     3181 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     3241 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     3301 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     3361 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     3421 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     3481 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     3541 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata
     3601 tggccacaac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     3661 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     3721 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     3781 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     3841 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     3901 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     3961 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     4021 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     4081 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     4141 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     4201 ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     4261 atgcatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4321 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4381 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4441 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4501 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc aagcttggcg
     4561 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4621 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4681 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     4741 cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4801 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4861 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4921 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     4981 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5041 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     5101 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5161 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5221 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5281 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5341 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5401 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5461 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5521 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5581 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5641 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5701 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5761 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5821 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5881 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5941 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6001 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6061 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6121 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6181 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6241 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6301 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6361 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6421 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6481 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6541 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6601 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6661 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6721 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6781 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6841 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6901 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atgaccgctt
     6961 tggaaaaaca aagactgtat ttcctggaaa ttaatgttta ttcaataaac tgtgtattca
     7021 gctatattca catagtggtg aggctgaaat gaggcgggaa gaggcggttg gggcttaatt
     7081 atatcaattt gggtggcccc acagcgcctc caaggcgcca gtcctgtttt gacaagttgc
     7141 ctctggaagc ctctcttctt tttctccaga gtaagcggag gccaggggcc cccggcctct
     7201 gcttaatact aaaaaaaac
//
