LOCUS       sequence-361590-        6230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for membrane-tethered 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 6230)
  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 6230)
  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-EGFP-TM-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..6230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    313..447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        478..977
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(513..536)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     910..929
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1049..1058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1118..1120,1121..1123,1124..1834)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    join(2040..2614,2615..2617,2618..2626)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2207..2224)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2434..2453
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2627..3226
                     /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(2627..2646)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3123..3143
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3307..3324)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3313..3537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3584..3600)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3584..3600)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3597..3619)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3608..3624
                     /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(3632..3638,3639..3656,3657..3662))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3677..3698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3757..3952
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3803..3938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3958..4092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4160..4177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4331..4919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4411..4430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5090..5881,5882..5950))
                     /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     5713..5732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaacagctgt tgtcatagta atgattgggt ggaaacattc caggcctggg tggagaggct
       61 ttttgcttcc tcttgcaaaa ccacactgac attccaggcc tgggtggaga ggctttttgc
      121 ttcctcttgc aaaaccacac tgccctctgg agggcagttg cctagcaact aattaaaaga
      181 ggatgtcgca cggccagctg cggtcagtta gtcacttcct gcttaactga cttgacattt
      241 tctattttaa gagtcgggag gaaaattact gtgttggagg ccctccgcca tcttctgaag
      301 ctgaatcgaa ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
      361 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
      421 atcaatgtat cttatcatgt ctggatctga tatcatcgtc gacctcgaaa ttctaccggg
      481 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
      541 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
      601 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
      661 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
      721 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
      781 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
      841 ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg
      901 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
      961 catctccggg cctttcgacc tgcatccatc tagatctcga gaattctcac gcgtcaagtg
     1021 gagcaaggca ggtggacagt ggatcgccgc caccatggag accgacaccc tcttgctctg
     1081 ggtcctgctg ctgtgggttc cgggtagtac cggagacatg gtttctaagg gtgaagaatt
     1141 gttcaccgga gttgtgccca tcctggttga gctggacgga gacgttaacg gacacaagtt
     1201 ctcagtgagc ggcgaaggag agggcgacgc tacgtacggt aaactgacgt tgaagttcat
     1261 ctgtacaaca ggaaagctgc cagtgccctg gcctacactc gtgaccaccc tcacctacgg
     1321 agtccagtgc ttctcccggt accccgacca catgaaacag cacgatttct tcaagtccgc
     1381 gatgcctgag ggctatgttc aggaacgcac catattcttc aaggacgacg gtaactataa
     1441 aaccagggct gaagttaagt tcgaggggga cacgttggta aatagaattg agttgaaagg
     1501 aatcgatttc aaagaagacg gcaatatttt gggccacaag ctggagtaca actacaacag
     1561 ccacaacgtg tatattatgg ctgataagca gaagaacggc atcaaggtga acttcaaaat
     1621 ccgacacaat atcgaggatg gctctgtgca actggccgac cattaccagc aaaacacgcc
     1681 tatcggcgac ggtccggttt tgctgccaga taaccactac ctctccacgc aatcagccct
     1741 ctccaaggat cctaacgaaa aaagagacca tatggttctg cttgaattcg tgacggctgc
     1801 tgggatcacc ctgggaatgg atgagctgta taagaacgcc gtgggccagg atacgcagga
     1861 ggtgatcgtc gtgccccaca gcttgccctt caaggtcgtg gttattagtg ctatcctggc
     1921 gttggtggtg ctgactatta tctcccttat catacttatc atgctgtggc aaaaaaaacc
     1981 ccgataagat ccttgacttg cggccgctcg ataagcttga tatcgaattc cgcccccccc
     2041 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     2101 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2161 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     2221 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     2281 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     2341 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     2401 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     2461 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     2521 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     2581 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatga ccgagtacaa
     2641 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     2701 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     2761 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     2821 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     2881 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     2941 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3001 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3061 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     3121 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3181 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     3241 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgatgatct agagctcgct
     3301 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3361 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3421 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3481 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3541 ctgaggcgga aagaacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg
     3601 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3661 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3721 ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat
     3781 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3841 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3901 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac
     3961 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4021 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4081 catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4141 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4201 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4261 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4321 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4381 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4441 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4501 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4561 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4621 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4681 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4741 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4801 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4861 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4921 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4981 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5041 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5101 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5161 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5221 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5281 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5341 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5401 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5461 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5521 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5581 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5641 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5701 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5761 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5821 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5881 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5941 gaatactcat actcttcctt tttcaatgac cgctttggaa aaacaaagac tgtatttcct
     6001 ggaaattaat gtttattcaa taaactgtgt attcagctat attcacatag tggtgaggct
     6061 gaaatgaggc gggaagaggc ggttggggct taattatatc aatttgggtg gccccacagc
     6121 gcctccaagg cgccagtcct gttttgacaa gttgcctctg gaagcctctc ttctttttct
     6181 ccagagtaag cggaggccag gggcccccgg cctctgctta atactaaaaa
//
