LOCUS       sequence-70881-e        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter driving GFP-IRES-Puromycin Acetyl Transferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  AUTHORS   Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith 
            A
  TITLE     Exit from pluripotency is gated by intracellular redistribution of 
            the bHLH transcription factor Tfe3.
  JOURNAL   Cell. 2013 Apr 11;153(2):335-47. doi: 10.1016/j.cell.2013.03.012.
  PUBMED    23582324
REFERENCE   2  (bases 1 to 7301)
  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"; doi: 
            "10.1016/j.cell.2013.03"; journalName: "Cell"; date: "2013-04-11- 
            11"; volume: "153"; issue: "2"; pages: "335-47"
COMMENT     Sequence Label: pPy-CAG-GFP-IRES-Pac
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        380..655
                     /label=chicken beta-actin promoter
     intron          656..1667
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1591..1613
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1675..1694
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1730..1754
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1760..1762,1763..1765,1766..2479)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1760..1762,1763..1765,1766..2476)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1805..1826)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2066..2085)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2413..2434
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2481..2505)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2639..3212,3213..3215,3216..3224)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2806..2823)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3033..3052
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3225..3824
                     /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(3225..3244)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3721..3741
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3905..3922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3911..4135
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4182..4198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4182..4198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4195..4217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4206..4222
                     /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(4230..4236,4237..4254,4255..4260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4275..4296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4355..4550
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4401..4536
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4556..4690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4758..4775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4929..5517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5009..5028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5688..6479,6480..6548))
                     /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     6311..6330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    7151..7285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        join(7299..7301,1..377)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      361 tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      421 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      481 ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg
      541 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
      601 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      661 cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct
      721 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
      781 taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa
      841 agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg
      901 tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc gctgcgggcg
      961 cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg gggcggtgcc
     1021 ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg
     1081 ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca cccccctccc
     1141 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg tggcgcgggg
     1201 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     1261 cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc ggctgtcgag
     1321 gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc
     1381 ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg
     1441 ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     1501 cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg
     1561 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     1621 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     1681 tgctggttat tgtgctgtct catcattttg gcaaagaatt cctcgaatca caagtttgta
     1741 caaaaaagca ggcttcacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1801 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1861 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1921 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1981 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2041 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2101 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2161 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2221 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2281 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2341 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2401 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2461 ggacgagctg tacaagtaag acccagcttt cttgtacaaa gtggtgattc gaggaattca
     2521 ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaac tggctcacaa
     2581 ataccactga gatcgacggt atcgagcggc cgctcgataa gcttgatatc gaattccgcc
     2641 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     2701 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     2761 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     2821 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     2881 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     2941 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     3001 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     3061 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     3121 catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga
     3181 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatgacc gagtacaagc
     3241 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     3301 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     3361 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3421 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3481 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3541 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3601 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3661 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3721 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3781 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     3841 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gatgatctag agctcgctga
     3901 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3961 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4021 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4081 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     4141 gaggcggaaa gaacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4201 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4261 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4321 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     4381 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4441 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4501 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     4561 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4621 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4681 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4741 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4801 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4861 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4921 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4981 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5041 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5101 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5161 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5221 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5281 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5341 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5401 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5461 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5521 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5581 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5641 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5701 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5761 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5821 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5881 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5941 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6001 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6061 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6121 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6181 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6241 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6301 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6361 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6421 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6481 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6541 atactcatac tcttcctttt tcaatgaccg ctttggaaaa acaaagactg tatttcctgg
     6601 aaattaatgt ttattcaata aactgtgtat tcagctatat cacatagtgg tgaggctgaa
     6661 atgaggcggg aagaggcggt tggggcttaa ttatatcaat ttgggtggcc ccacagcgcc
     6721 tccaaggcgc cagtcctgtt ttgacaagtt gcctctggaa gcctctacaa tgcctctctt
     6781 ctttttctcc agagtaagcg gaggccaggg gcccccggcc tctgcttaat actaaaaaaa
     6841 acagctgttg tcatagtaat gattgggtgg aaacattcca ggcctgggtg gagaggcttt
     6901 ttgcttcctc ttgcaaaacc acactgacat tccaggcctg ggtggagagg ctttttgctt
     6961 cctcttgcaa aaccacactg ccctctggag ggcagttgcc tagcaactaa ctaaaagagg
     7021 atgtcgcacg gccagctgcg gtcagttagt cacttcctgc ttaactgact tgacattttc
     7081 tattttaaga gtcgggagga aaattactgt gttggaggcc ctccgccatc ttctgaagct
     7141 gaatcgaatt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7201 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7261 caatgtatct tatcatgtct ggatctgata tcatcgtcga c
//
