LOCUS       sequence-317505-        6186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6186)
  AUTHORS   Lukow DA, Sausville EL, Suri P, Chunduri NK, Wieland A, Leu J, Smith
            JC, Girish V, Kumar AA, Kendall J, Wang Z, Storchova Z, Sheltzer JM
  TITLE     Chromosomal instability accelerates the evolution of resistance to 
            anti-cancer therapies.
  JOURNAL   Dev Cell. 2021 Sep 13;56(17):2427-2439.e4. doi: 
            10.1016/j.devcel.2021.07.009. Epub 2021 Aug 4.
  PUBMED    34352222
REFERENCE   2  (bases 1 to 6186)
  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.devcel.2021.07.009"; journalName: "Dev Cell"; date: 
            "2021-09-13- 13"; volume: "56"; issue: "17"; pages: "2427-2439.e4"
COMMENT     Sequence Label: pMMLV-EGFP-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..6186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..594
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    657..1014
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1073..1489
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1419..1441
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1457..1473
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1499..1873
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1811..1827
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     protein_bind    1924..1948
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1949..1958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1955..1957,1958..1960,1961..2671)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1955..1957,1958..1960,1961..2671)
                     /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(2000..2021)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2261..2280)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2608..2629
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2672..2728
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2729..3328
                     /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(2729..2748)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3225..3245
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(3329..3353)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     LTR             3670..4139
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4269..4286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4440..5028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4520..4539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5192..5983,5984..6052))
                     /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     5815..5834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6053..6157)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac
      121 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      181 aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg
      241 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct
      301 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
      361 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
      421 ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact gagtcgcccg
      481 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
      541 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg
      601 ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
      661 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc
      721 ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
      781 gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt
      841 tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg caccccccta
      901 ataggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc
      961 tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat
     1021 cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt agggccagac
     1081 tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc ggatcgctca
     1141 caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg cagaatggcc
     1201 aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca tcacccaggt
     1261 taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc cctacatcgt
     1321 gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg tacaccctaa
     1381 gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc ctcgttcgac
     1441 cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccc ccatatggcc
     1501 atatgagatc ttatatgggg cacccccgcc ccttgtaaac ttccctgacc ctgacatgac
     1561 aagagttact aacagcccct ctctccaagc tcacttacag gctctctact tagtccagca
     1621 cgaagtctgg agacctctgg cggcagccta ccaagaacaa ctggaccgac cggtggtacc
     1681 tcacccttac cgagtcggcg acacagtgtg ggtccgccga caccagacta agaacctaga
     1741 acctcgctgg aaaggacctt acacagtcct gctgaccacc cccaccgccc tcaaagtaga
     1801 cggcatcgca gcttggatac acgccgccca cgtgaaggct gccgaccccg ggggtggacc
     1861 atcctctaga ctgccggatc tagctagtta attaaggatc ccagtgtggt ggtacgggaa
     1921 atcacaagtt tgtacaaaaa agcaggctgc caccatggtg agcaagggcg aggagctgtt
     1981 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2041 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2101 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2161 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2221 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2281 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2341 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2401 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2461 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2521 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2581 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2641 gatcactctc ggcatggacg agctgtacaa ggccacgaac ttctctctgt taaagcaagc
     2701 aggagatgtt gaagaaaacc ccgggcctat gaccgagtac aagcccacgg tgcgcctcgc
     2761 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2821 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     2881 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     2941 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3001 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3061 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     3121 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3181 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     3241 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3301 gtgcatgacc cgcaagcccg gtgcctgaac ccagctttct tgtacaaagt ggtgatattc
     3361 cagctgagcg ccggtcgcta ccattaccag ttggtctggt gtcaaaaata ataataaccg
     3421 ggcaggccat gtctgcccgt atttcgcgta aggaaatcca ttatgtacta tttaaactcg
     3481 agcggccgcc agcacagtgg tcgacgataa aataaaagat tttatttagt ctccagaaaa
     3541 aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt
     3601 gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg tcaggaacag
     3661 atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     3721 cagggccaag aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     3781 gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc agccctcagc
     3841 agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg
     3901 ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc
     3961 gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag
     4021 tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc
     4081 gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcac
     4141 atgcagcatg tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca
     4201 tagttgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4261 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4321 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4381 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4441 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4501 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4561 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4621 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4681 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4741 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4801 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4861 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4921 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4981 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5041 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5101 atgagattat caaaaaggat cttcacctag atccttttgc ggccggccgc aaatcaatct
     5161 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5221 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5281 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5341 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5401 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5461 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5521 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5581 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5641 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5701 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5761 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5821 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5881 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5941 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6001 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6061 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6121 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6181 ctgcat
//
