LOCUS       sequence-237315-        5329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMVP L3-L2 entry plasmid, contains HA tag-polyA + EF1a-TETa-polyA 
            for 3- or 4-component MultiSite Gateway Pro assembly. Adds C-term HA
            tag to gene plus downstream EF1a-driven TETa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5329)
  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 5329)
  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: KO701: pMVP (L3-L2) HA tag + pA; EF1a::TETa-pA
FEATURES             Location/Qualifiers
     source          1..5329
                     /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"
     promoter        944..2122
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1175..2113
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2070..2090
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2139..2157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2199..2816,2817..2822,2823..2858,2859..2861,2862..2897,
                     2898..2900,2901..2936,2937..2945)
                     /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    2968..3089
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2979..2998)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3441..3540)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3571..3589)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3594..3610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3594..3610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3712..3731)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3723..4532
                     /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(3790..3809)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4679..5267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5168..5187
                     /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 agtctagacg tgaggctccg gtgcccgtca
      961 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     1021 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     1081 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     1141 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
     1201 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
     1261 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     1321 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     1381 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     1441 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     1501 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     1561 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     1621 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     1681 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     1741 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     1801 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     1861 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     1921 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     1981 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     2041 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     2101 tcttccattt caggtgtcgt gaggaattag cttggtacta atacgactca ctatagggag
     2161 acccaagctg gctaggtgaa ttcgactggc gcgccaccat gtccagactg gacaagagca
     2221 aagtcataaa cggcgctctg gaattactca atggagtcgg tatcgaaggc ctgacgacaa
     2281 ggaaactcgc tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca
     2341 agcgggccct gctcgatgcc ctgccaatcg agatgctgga caggcatcat acccacttct
     2401 gccccctgga aggcgagtca tggcaagact ttctgcggaa caacgccaag tcattccgct
     2461 gtgctctcct ctcacatcgc gacggggcta aagtgcatct cggcacccgc ccaacagaga
     2521 aacagtacga aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg
     2581 agaacgcact gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg
     2641 aacaggagca tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc
     2701 cacttctgag acaagcaatt gagctgttcg accggcaggg agccgaacct gccttccttt
     2761 tcggcctgga actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc
     2821 cggccgacgc ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact
     2881 ttgaccttga tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg
     2941 ggtaactaag taagaatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     3001 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     3061 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3121 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     3181 tggctgatta tgatcctgca agcctcgtcg tcctggccgg accacgctat ctgtgcaagg
     3241 tccccggccc cggacgcgcg ctccatgagc agagcgcccg ccgccgaggc gaagactcgg
     3301 gcggcgccct gcccgtccca ccaggtcaac aggcggtaac cggcctcttc atcgggaatg
     3361 cgcgcgacct tcagcatcgc cggcatgtcc ccctggcgga cgggaagtat ccagctcgac
     3421 caggcttgtc gactcgaaga acccagcttt cttgtacaaa gttggcatta taagaaagca
     3481 ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg
     3541 ccatccagct gcagggcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag
     3601 ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata
     3661 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     3721 ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg
     3781 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     3841 gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     3901 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     3961 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     4021 ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     4081 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     4141 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     4201 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     4261 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     4321 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     4381 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     4441 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     4501 agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact
     4561 ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     4621 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4681 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4741 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4801 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4861 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4921 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4981 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5041 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5101 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5161 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5221 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5281 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
