LOCUS       sequence-361589-        7425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for membrane-tethered EGFP from the CAG 
            promoter. Confers puromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7425)
  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 7425)
  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-EGFP-TM-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..7425
                     /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        839..1114
                     /label=chicken beta-actin promoter
     intron          1115..2123
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2047..2069
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2131..2150
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2231..2240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2300..2302,2303..2305,2306..3016)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    join(3222..3795,3796..3798,3799..3807)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3389..3406)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3616..3635
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3808..4407
                     /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(3808..3827)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4304..4324
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4488..4505)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4494..4718
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4765..4781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4765..4781)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4778..4800)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4789..4805
                     /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(4813..4819,4820..4837,4838..4843))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4858..4879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4938..5133
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4984..5119
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5139..5273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5341..5358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5512..6100)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5592..5611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6271..7062,7063..7131))
                     /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     6894..6913
                     /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 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      721 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      781 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatgggtc
      841 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      901 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg ggggggggcg
      961 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
     1021 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     1081 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgttgc cttcgccccg
     1141 tgccccgctc cgcgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
     1201 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg
     1261 acggctcgtt tcttttctgt ggctgcgtga aagccttaaa gggctccggg agggcccttt
     1321 gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt
     1381 gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     1441 ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg gggggctgcg
     1501 aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg
     1561 cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc gagttgctga gcacggcccg
     1621 gcttcgggtg cggggctccg tgcggggcgt ggcgcggggc tcgccgtgcc gggcgggggg
     1681 tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg
     1741 gaggggcgcg gcggccccgg agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt
     1801 gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctggcgga
     1861 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcgggcgaa gcggtgcggc
     1921 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     1981 ctccatctcc agcctcgggg ctgccgcagg gggacggctg ccttcggggg ggacggggca
     2041 gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc
     2101 atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc
     2161 attttggcaa agaattcctc gagaattctc acgcgtcaag tggagcaagg caggtggaca
     2221 gtggatcgcc gccaccatgg agaccgacac cctcttgctc tgggtcctgc tgctgtgggt
     2281 tccgggtagt accggagaca tggtttctaa gggtgaagaa ttgttcaccg gagttgtgcc
     2341 catcctggtt gagctggacg gagacgttaa cggacacaag ttctcagtga gcggcgaagg
     2401 agagggcgac gctacgtacg gtaaactgac gttgaagttc atctgtacaa caggaaagct
     2461 gccagtgccc tggcctacac tcgtgaccac cctcacctac ggagtccagt gcttctcccg
     2521 gtaccccgac cacatgaaac agcacgattt cttcaagtcc gcgatgcctg agggctatgt
     2581 tcaggaacgc accatattct tcaaggacga cggtaactat aaaaccaggg ctgaagttaa
     2641 gttcgagggg gacacgttgg taaatagaat tgagttgaaa ggaatcgatt tcaaagaaga
     2701 cggcaatatt ttgggccaca agctggagta caactacaac agccacaacg tgtatattat
     2761 ggctgataag cagaagaacg gcatcaaggt gaacttcaaa atccgacaca atatcgagga
     2821 tggctctgtg caactggccg accattacca gcaaaacacg cctatcggcg acggtccggt
     2881 tttgctgcca gataaccact acctctccac gcaatcagcc ctctccaagg atcctaacga
     2941 aaaaagagac catatggttc tgcttgaatt cgtgacggct gctgggatca ccctgggaat
     3001 ggatgagctg tataagaacg ccgtgggcca ggatacgcag gaggtgatcg tcgtgcccca
     3061 cagcttgccc ttcaaggtcg tggttattag tgctatcctg gcgttggtgg tgctgactat
     3121 tatctccctt atcatactta tcatgctgtg gcaaaaaaaa ccccgataag atccttgact
     3181 tgcggccgct cgataagctt gatatcgaat tccgcccccc ccccctctcc ctcccccccc
     3241 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3301 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3361 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3421 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     3481 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     3541 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3601 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3661 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3721 tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     3781 aaacacgatg ataatatggc cacaaccatg accgagtaca agcccacggt gcgcctcgcc
     3841 acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     3901 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3961 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4021 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4081 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4141 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     4201 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     4261 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     4321 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     4381 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa
     4441 aggagcgcac gaccccatgc atcgatgatc tagagctcgc tgatcagcct cgactgtgcc
     4501 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4561 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4621 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4681 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaacctg
     4741 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4801 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4861 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4921 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4981 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     5041 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     5101 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     5161 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5221 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     5281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5341 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5401 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5461 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5521 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5581 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5641 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5701 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5761 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5821 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5881 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5941 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6001 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6061 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6121 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6181 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6241 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6301 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6361 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6421 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6481 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6541 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6601 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6661 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6721 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6781 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6841 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6901 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6961 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7021 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7081 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7141 ttttcaatga ccgctttgga aaaacaaaga ctgtatttcc tggaaattaa tgtttattca
     7201 ataaactgtg tattcagcta tatcacatag tggtgaggct gaaatgaggc gggaagaggc
     7261 ggttggggct taattatatc aatttgggtg gccccacagc gcctccaagg cgccagtcct
     7321 gttttgacaa gttgcctctg gaagcctcta caatgcctct cttctttttc tccagagtaa
     7381 gcggaggcca ggggcccccg gcctctgctt aatactaaaa aaaac
//
