LOCUS       sequence-237308-        5491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMVP L3-L2 entry plasmid, contains HA tag-polyA + 
            CMV-eGFP-P2A-TETa-polyA for 3- or 4-component MultiSite Gateway Pro 
            assembly. Adds C-term HA tag to gene plus downstream CMV-driven 
            GFP-P2A-TETa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5491)
  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 5491)
  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: KA801: pMVP (L3-L2) HA tag + pA;
            CMV::eGFP-P2A...
FEATURES             Location/Qualifiers
     source          1..5491
                     /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             686..712
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    845..926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        990..1293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1294..1497
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1448..1468
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1563..1565,1566..1568,1569..2279)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1563..1565,1566..1568,1569..2279)
                     /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(1869..1888)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2216..2237
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2295..2351
                     /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(2361..2978,2979..2984,2985..3020,3021..3023,3024..3059,
                     3060..3062,3063..3098,3099..3107)
                     /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    3130..3251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3141..3160)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3603..3702)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3732..3751)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3733..3751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3756..3772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3756..3772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3874..3893)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3885..4694
                     /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(3952..3971)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4841..5429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5330..5349
                     /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 aacaaagctg gatcctaccc atacgatgtg ccagattacg cttgatttaa
      721 atagataact gactagagga tcataatcag ccataccaca tttgtagagg ttttacttgc
      781 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
      841 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
      901 cacaaataaa gcattttttt cactgcctcg agtctagaat agtaatcaat tacggggtca
      961 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     1021 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1081 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1141 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     1201 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     1261 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     1321 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     1381 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     1441 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     1501 ttagtgaacc gtcagatccg ctagcgctac cggactcaga tctcgaattc gactggcgcg
     1561 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1621 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1681 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1741 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1801 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1861 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1921 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1981 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2041 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2101 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2161 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2221 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagc
     2281 gcgccggatc aggagctact aatttctctc tgttaaagca ggcaggagat gtggaggaga
     2341 atccaggtcc tcaattcacc atgtccagac tggacaagag caaagtcata aacggcgctc
     2401 tggaattact caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc
     2461 tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg
     2521 ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt
     2581 catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc
     2641 gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg
     2701 aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc
     2761 tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag
     2821 caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa
     2881 ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca
     2941 tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg
     3001 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     3061 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaacta agtaagaatc
     3121 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3181 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3241 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     3301 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctg
     3361 caagcctcgt cgtcctggcc ggaccacgct atctgtgcaa ggtccccggc cccggacgcg
     3421 cgctccatga gcagagcgcc cgccgccgag gcgaagactc gggcggcgcc ctgcccgtcc
     3481 caccaggtca acaggcggta accggcctct tcatcgggaa tgcgcgcgac cttcagcatc
     3541 gccggcatgt ccccctggcg gacgggaagt atccagctcg accaggcttg tcgactcgaa
     3601 gaacccagct ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg
     3661 caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagggcg
     3721 gccgcgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct
     3781 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     3841 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     3901 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     3961 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     4021 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     4081 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     4141 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     4201 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     4261 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     4321 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     4381 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     4441 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     4501 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     4561 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     4621 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     4681 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga
     4741 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     4801 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4861 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4921 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4981 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5041 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5101 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5161 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5221 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5281 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5341 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5401 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5461 tggccttttg ctggcctttt gctcacatgt t
//
