LOCUS       sequence-361591-        7511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for membrane-tethered HA- and Myc-tagged
            EGFP from the CAG promoter. Confers puromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7511)
  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 7511)
  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: pPyCAG-HA-EGFP-Myc-TM-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..7511
                     /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"
     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      2194..2203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     2200..2259
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             2263..2289
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2362..2371
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2368..2370,2371..2373,2374..3084)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2368..2370,2371..2373,2374..3084)
                     /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(2413..2434)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2674..2693)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3021..3042
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3094..3123
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3127..3273
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     misc_feature    join(3317..3891,3892..3894,3895..3903)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3484..3501)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3711..3730
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3904..4503
                     /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(3904..3923)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4400..4420
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4584..4601)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4590..4814
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4861..4877)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4861..4877)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4874..4896)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4885..4901
                     /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(4909..4915,4916..4933,4934..4939))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4954..4975
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5034..5229
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5080..5215
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5235..5369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5437..5454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5608..6196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5688..5707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6367..7158,7159..7227))
                     /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     6990..7009
                     /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 sgaggcggag 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 grgggctcgg
     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 ttcctcgagt gccgccacca tggagacaga cacactcctg
     2221 ctatgggtac tgctgctctg ggttccaggt tccactggtg actatccata tgatgttcca
     2281 gattatgctg gggcccagcc ggccagatct cccgggatcc gcgggtttag tgaaccgtca
     2341 gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     2401 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2461 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2521 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2581 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2641 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2701 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2761 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2821 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2881 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2941 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     3001 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     3061 ctcggcatgg acgagctgta caagtccgtc gacgaacaaa aactcatctc agaagaggat
     3121 ctgaatgctg tgggccagga cacgcaggag gtcatcgtgg tgccacactc cttgcccttt
     3181 aaggtggtgg tgatctcagc catcctggcc ctggtggtgc tcaccatcat ctcccttatc
     3241 atcctcatca tgctttggca gaagaagcca cgttaggcgg ccgctcgata agcttgatat
     3301 cgaattccgc cccccccccc tctccctccc ccccccctaa cgttactggc cgaagccgct
     3361 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     3421 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     3481 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     3541 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     3601 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     3661 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     3721 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     3781 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     3841 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca
     3901 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3961 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4021 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4081 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4141 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4201 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     4261 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4321 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     4381 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4441 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4501 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     4561 atgatctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4621 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4681 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4741 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4801 ggtgggctct atggcttctg aggcggaaag aacctgcagc ccaagcttgg cgtaatcatg
     4861 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4921 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4981 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc
     5041 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5101 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5161 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5221 ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5281 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5341 tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg
     5401 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5461 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5521 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5581 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5641 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5701 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5761 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5821 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5881 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5941 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6001 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6061 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6121 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6181 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6241 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6301 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6361 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6421 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6481 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6541 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6601 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6661 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6721 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6781 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6841 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6901 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6961 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7021 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7081 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7141 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7201 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatgaccgc tttggaaaaa
     7261 caaagactgt atttcctgga aattaatgtt tattcaataa actgtgtatt cagctatatt
     7321 cacatagtgg tgaggctgaa atgaggcggg aagaggcggt tggggcttaa ttatatcaat
     7381 ttgggtggcc ccacagcgcc tccaaggcgc cagtcctgtt ttgacaagtt gcctctggaa
     7441 gcctctcttc tttttctcca gagtaagcgg aggccagggg cccccggcct ctgcttaata
     7501 ctaaaaaaaa c
//
