LOCUS       sequence-408699-        7739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express MMLV Gag-P2A-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7739)
  AUTHORS   Horns F, Martinez JA, Fan C, Haque M, Linton JM, Tobin V, Santat L, 
            Maggiolo AO, Bjorkman PJ, Lois C, Elowitz MB
  TITLE     Engineering RNA export for measurement and manipulation of living 
            cells.
  JOURNAL   Cell. 2023 Jul 10:S0092-8674(23)00689-X. doi: 
            10.1016/j.cell.2023.06.013.
  PUBMED    37437570
REFERENCE   2  (bases 1 to 7739)
  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: "Cell. 2023 
            Jul 10:S0092-8674(23)00689-X. doi: 10.1016/j.cell.2023.06.013."
COMMENT     Sequence Label: pFH2.9_MMLV_Gag-P2A-eGFP
FEATURES             Location/Qualifiers
     source          1..7739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      923..932
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2531..2587
                     /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             join(2588..2590,2591..3304)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2588..3301
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2630..2651)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3238..3259
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3330..3347)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3336..3560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3606..4034
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3693..3712)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3903..3924
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4043..4063)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4048..4377
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4228..4363
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4290..4309
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4444..5238
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4498..4517)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5108..5127
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5412..5533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5449..5468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5503..5522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5582..5598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5582..5598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5595..5617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5606..5622
                     /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(5630..5636,5637..5654,5655..5660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5675..5696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5813..5830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5984..6572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6064..6083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6743..7534,7535..7603))
                     /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     7366..7385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7604..7708)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgccaccat gggccagacc gtgaccaccc ctctgagcct
      961 gaccctgggt cattggaagg acgtcgagcg gatcgcccac aaccagagcg tggacgtgaa
     1021 aaagagacgg tgggtgacct tctgcagcgc tgagtggcct accttcaacg tgggctggcc
     1081 tcgggatggc acctttaata gggatctgat tacccaggtt aagatcaagg tgttctcccc
     1141 tggcccccac ggccaccccg accaggtgcc ttacatcgtg acatgggagg ctctcgcctt
     1201 cgaccctcct ccttgggtta agcctttcgt gcaccctaag cccccccctc ctctgcctcc
     1261 atcagctcca agcctgcccc tggaacctcc ccggtccacc ccccccagaa gcagcctgta
     1321 tccagctctg accccttctc tgggcgccaa gccaaagccc caagtgctga gcgatagcgg
     1381 cggacctctg atcgacctgc tgaccgagga ccctcctcct tacagggacc ccaggcctcc
     1441 tccttctgac agagacggca acggcggcga ggccacacct gccggcgagg cccctgaccc
     1501 ctccccaatg gccagcagac tgcggggcag aagagaaccc cctgtggccg acagcaccac
     1561 tagccaggcc tttcctctga gagccggagg caatggccaa ctgcagtact ggcccttcag
     1621 ctcttctgat ctgtacaact ggaaaaacaa caatccttct ttcagcgagg accctggcaa
     1681 gctgaccgcc ctgatcgaga gcgtgctgat cacccaccag cctacatggg atgattgcca
     1741 gcagctgctg ggaacactgc tcaccggcga agagaagcaa agagtgctgc tcgaggccag
     1801 aaaggccgtg cgaggcgacg acggcagacc tacacagctg cctaacgagg tggacgccgc
     1861 cttccccctg gaaagacccg actgggacta caccacacag gccggcagaa accacctggt
     1921 ccactaccgg cagcttctgc tggcaggcct gcagaacgcc ggcagatctc caacaaacct
     1981 ggctaaagtg aagggcatca cacaaggccc taatgagagc cccagcgcct ttctggaaag
     2041 actgaaagaa gcctaccgga gatatacacc ctacgacccc gaggaccctg gacaggagac
     2101 caacgtgtct atgagcttca tctggcagag cgctcctgac atcggccgca agctggaacg
     2161 cctcgaggac ctgaagaaca agaccctggg cgatctggtg cgggaagccg agaaaatctt
     2221 caacaagcgg gaaacccctg aggaacggga agagagaatc agacgggaga cagaggaaaa
     2281 ggaagagaga agaagaaccg aagatgagca gaaggaaaag gagagagata gacggcggca
     2341 cagagaaatg agcaagctgc tggccaccgt ggtgtccgga cagaaacagg acagacaggg
     2401 cggagagaga cggcggagcc agctggacag agatcaatgt gcctactgca aggagaaagg
     2461 ccactgggcc aaggactgcc ctaagaagcc cagaggacct agaggcccta gacctcagac
     2521 atccctgctg gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     2581 tggacctatg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2641 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     2701 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2761 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     2821 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2881 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2941 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3001 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     3061 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3121 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3181 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     3241 ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg agctgtacaa
     3301 gtaagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3541 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3601 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3661 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3721 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3781 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3841 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3901 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3961 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4021 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4081 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4141 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4201 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4261 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     4321 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4381 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     4441 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4501 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4561 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4621 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4681 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4741 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4801 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4861 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4921 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4981 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     5041 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     5101 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     5161 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     5221 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     5281 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     5341 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     5401 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5461 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5521 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5581 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5641 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5701 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5821 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5881 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5941 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6001 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6061 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6121 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6181 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6241 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6301 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6361 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6421 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6481 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6541 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6601 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6661 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6721 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6781 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6841 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6901 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6961 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7021 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7081 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7141 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7201 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7261 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7321 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7381 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7441 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7501 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7561 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7621 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7681 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
