LOCUS       sequence-237312-        6099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMVP L3-L2 entry plasmid, contains polyA + EF1a-eGFP-P2A-TETa-polyA 
            for 3- or 4-component MultiSite Gateway Pro assembly. Adds 
            EF1a-driven GFP-P2A-TETa downstream of gene of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6099)
  AUTHORS   Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, 
            Newgard CB
  TITLE     Creation of versatile cloning platforms for transgene expression and
            dCas9-based epigenome editing.
  JOURNAL   Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 
            10.1093/nar/gky1286.
  PUBMED    30590691
REFERENCE   2  (bases 1 to 6099)
  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: "Nucleic 
            Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286."
COMMENT     Sequence Label: JN511: pMVP (L3-L2)  pA; EF1a::eGFP-P2A-TETa-pA
FEATURES             Location/Qualifiers
     source          1..6099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    570..665
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    820..901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        919..2097
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1150..2088
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2045..2065
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2114..2132
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2171..2173,2174..2176,2177..2887)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2171..2173,2174..2176,2177..2887)
                     /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(2477..2496)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2824..2845
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2903..2959
                     /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(2969..3586,3587..3592,3593..3628,3629..3631,3632..3667,
                     3668..3670,3671..3706,3707..3715)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    3738..3859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3749..3768)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4211..4310)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4341..4359)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4364..4380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4364..4380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4482..4501)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4493..5302
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4560..4579)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5449..6037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5938..5957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtataataa
      661 agttgagatc aacaaagctt cctagctcga tttaaataga taactgacta gaggatcata
      721 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
      781 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
      841 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      901 cctcgagtct agacgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      961 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     1021 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     1081 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1141 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     1201 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     1261 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     1321 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     1381 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     1441 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     1501 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     1561 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     1621 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     1681 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     1741 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     1801 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     1861 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     1921 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     1981 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     2041 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagga
     2101 attagcttgg tactaatacg actcactata gggagaccca agctggctag gtgaattcga
     2161 ctggcgcgcc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2221 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2281 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2341 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2401 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2461 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2521 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2581 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2641 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2701 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2761 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2821 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2881 gtacaagcgc gccggatcag gagctactaa tttctctctg ttaaagcagg caggagatgt
     2941 ggaggagaat ccaggtcctc aattcaccat gtccagactg gacaagagca aagtcataaa
     3001 cggcgctctg gaattactca atggagtcgg tatcgaaggc ctgacgacaa ggaaactcgc
     3061 tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct
     3121 gctcgatgcc ctgccaatcg agatgctgga caggcatcat acccacttct gccccctgga
     3181 aggcgagtca tggcaagact ttctgcggaa caacgccaag tcattccgct gtgctctcct
     3241 ctcacatcgc gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga
     3301 aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact
     3361 gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg aacaggagca
     3421 tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgag
     3481 acaagcaatt gagctgttcg accggcaggg agccgaacct gccttccttt tcggcctgga
     3541 actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cggccgacgc
     3601 ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact ttgaccttga
     3661 tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg ggtaactaag
     3721 taagaatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3781 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3841 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3901 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     3961 tgatcctgca agcctcgtcg tcctggccgg accacgctat ctgtgcaagg tccccggccc
     4021 cggacgcgcg ctccatgagc agagcgcccg ccgccgaggc gaagactcgg gcggcgccct
     4081 gcccgtccca ccaggtcaac aggcggtaac cggcctcttc atcgggaatg cgcgcgacct
     4141 tcagcatcgc cggcatgtcc ccctggcgga cgggaagtat ccagctcgac caggcttgtc
     4201 gactcgaaga acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     4261 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     4321 gcagggcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     4381 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     4441 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     4501 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     4561 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     4621 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     4681 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     4741 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     4801 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     4861 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     4921 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     4981 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5041 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5101 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5161 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5221 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5281 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     5341 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     5401 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5461 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5521 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5581 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5641 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5701 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5761 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5821 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5881 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5941 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6001 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6061 acggttcctg gccttttgct ggccttttgc tcacatgtt
//
