LOCUS       sequence-361592-        6319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for membrane-tethered HA- and Myc-tagged
            EGFP from the mouse Pgk1 promoter. Confers puromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6319)
  AUTHORS   Malaguti M, Migueles RP, Annoh J, Sadurska D, Blin G, Lowell S
  TITLE     SyNPL: Synthetic notch pluripotent cell lines to monitor and 
            manipulate cell interactions in vitro and in vivo.
  JOURNAL   Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226.
  PUBMED    35616331
REFERENCE   2  (bases 1 to 6319)
  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: 
            "Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226."
COMMENT     Sequence Label: pPyPGK-HA-EGFP-Myc-TM-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..6319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    353..487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        518..1017
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(553..576)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     950..969
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1046..1055
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     1052..1111
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             1115..1141
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1214..1223
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1220..1222,1223..1225,1226..1936)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1220..1222,1223..1225,1226..1936)
                     /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(1265..1286)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1526..1545)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1873..1894
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1946..1975
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1979..2125
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     misc_feature    join(2169..2743,2744..2746,2747..2755)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2336..2353)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2563..2582
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2756..3355
                     /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(2756..2775)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3252..3272
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3436..3453)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3442..3666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3713..3729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3713..3729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3726..3748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3737..3753
                     /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(3761..3767,3768..3785,3786..3791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3806..3827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3886..4081
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3932..4067
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4087..4221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4289..4306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4460..5048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4540..4559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5219..6010,6011..6079))
                     /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     5842..5861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cggaggccag gggcccccgg cctctgctta atactaaaaa aaacagctgt tgtcatagta
       61 atgattgggt ggaaacattc caggcctggg tggagaggct ttttgcttcc tcttgcaaaa
      121 ccacactgac attccaggcc tgggtggaga ggctttttgc ttcctcttgc aaaaccacac
      181 tgccctctgg agggcagttg cctagcaact aattaaaaga ggatgtcgca cggccagctg
      241 cggtcagtta gtcacttcct gcttaactga cttgacattt tctattttaa gagtcgggag
      301 gaaaattact gtgttggagg ccctccgcca tcttctgaag ctgaatcgaa ttaacttgtt
      361 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
      421 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
      481 ctggatctga tatcatcgtc gacctcgaaa ttctaccggg taggggaggc gcttttccca
      541 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
      601 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
      661 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
      721 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
      781 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
      841 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
      901 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
      961 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     1021 tgcatccatc tagatctcga gtgccgccac catggagaca gacacactcc tgctatgggt
     1081 actgctgctc tgggttccag gttccactgg tgactatcca tatgatgttc cagattatgc
     1141 tggggcccag ccggccagat ctcccgggat ccgcgggttt agtgaaccgt cagatccgct
     1201 agcgctaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1261 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1321 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1381 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1441 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1501 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1561 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1621 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1681 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1741 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1801 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1861 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1921 ggacgagctg tacaagtccg tcgacgaaca aaaactcatc tcagaagagg atctgaatgc
     1981 tgtgggccag gacacgcagg aggtcatcgt ggtgccacac tccttgccct ttaaggtggt
     2041 ggtgatctca gccatcctgg ccctggtggt gctcaccatc atctccctta tcatcctcat
     2101 catgctttgg cagaagaagc cacgttaggc ggccgctcga taagcttgat atcgaattcc
     2161 gccccccccc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     2221 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     2281 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     2341 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     2401 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     2461 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     2521 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     2581 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     2641 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     2701 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatgac
     2761 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2821 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     2881 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2941 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3001 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3061 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3121 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3181 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3241 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3301 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc
     3361 gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cgatgatcta
     3421 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3481 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3541 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3601 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3661 ctatggcttc tgaggcggaa agaacctgca gcccaagctt ggcgtaatca tggtcatagc
     3721 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3781 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc
     3901 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3961 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4021 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4081 aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4141 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4201 gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4261 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4321 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4381 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4441 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4501 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4561 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4621 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4681 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4741 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4801 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4861 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4921 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4981 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5041 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5101 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5161 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5221 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5281 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5341 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5401 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5461 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5521 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5581 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5641 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5701 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5761 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5821 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5881 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5941 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6001 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6061 ggaaatgttg aatactcata ctcttccttt ttcaatgacc gctttggaaa aacaaagact
     6121 gtatttcctg gaaattaatg tttattcaat aaactgtgta ttcagctata ttcacatagt
     6181 ggtgaggctg aaatgaggcg ggaagaggcg gttggggctt aattatatca atttgggtgg
     6241 ccccacagcg cctccaaggc gccagtcctg ttttgacaag ttgcctctgg aagcctctct
     6301 tctttttctc cagagtaag
//
