LOCUS       sequence-237186-        4193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMVP L3-L2 entry plasmid, contains OLLAS epitope tag + IRES2-eGFP + 
            polyA for 3- or 4-component MultiSite Gateway Pro assembly. Allows 
            C-term epitope fusion and IRES expression of eGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4193)
  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 4193)
  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: IX702: pMVP (L3-L2) OLLAS-IRES-eGFP + polyA
FEATURES             Location/Qualifiers
     source          1..4193
                     /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"
     CDS             677..718
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     misc_feature    722..774
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(776..1350,1351..1353,1354..1362)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(943..960)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1170..1189
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1363..1365,1366..1368,1369..2082)
                     /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(1363..1365,1366..1368,1369..2079)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1408..1429)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1669..1688)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2016..2037
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2205..2286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2242..2261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2305..2404)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2434..2453)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2435..2453)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2458..2474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2458..2474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2576..2595)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2587..3396
                     /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(2654..2673)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3543..4131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4032..4051
                     /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 agttgtagct agcggaagcg gcttcgccaa cgagctggga cccaggctga tgggaaagta
      721 actcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccgcccct
      781 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
      841 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
      901 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
      961 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     1021 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     1081 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     1141 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     1201 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat
     1261 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     1321 tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatggtgag caagggcgag
     1381 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1441 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1501 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     1561 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1621 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1681 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1741 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1801 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1861 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1921 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1981 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2041 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga
     2101 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2161 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2221 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2281 cactgcattc tagttgtggt ttgaacccag ctttcttgta caaagttggc attataagaa
     2341 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     2401 tttgccatcc agctgcaggg cggccgcgat atcccctata gtgagtcgta ttacatggtc
     2461 atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa
     2521 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     2581 ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat
     2641 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     2701 tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     2761 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     2821 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     2881 atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     2941 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     3001 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     3061 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     3121 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     3181 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     3241 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     3301 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     3361 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     3421 cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt
     3481 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3541 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3601 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3661 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     3721 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3781 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3841 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3901 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3961 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4021 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4081 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4141 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
