LOCUS       Exported                6124 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMVP L3-L2 entry plasmid, contains HA tag-polyA + 
            EF1a-eGFP-P2A-TETa-polyA for 3- or 4-component MultiSite Gateway Pro
            assembly. Adds C-term HA tag to gene plus downstream EF1a-driven 
            GFP-P2A-TETa.
ACCESSION   .
VERSION     .
KEYWORDS    KA901: pMVP (L3-L2) HA tag + pA; EF1a::eGFP-P2A-TETa-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6124)
  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 6124)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6124
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      268..295
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      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             2196..2912
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2196..2912
                     /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(2241..2262)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2502..2521)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2849..2870
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2928..2984
                     /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             2994..3740
                     /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    3763..3884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3774..3793)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4236..4335)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4366..4384)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4389..4405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4389..4405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4507..4526)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4518..5327
                     /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(4585..4604)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5474..6062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5963..5982
                     /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 gcgccatggt gagcaagggc gaggagctgt
     2221 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     2281 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2341 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     2401 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2461 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2521 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2581 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2641 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     2701 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2761 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2821 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2881 ggatcactct cggcatggac gagctgtaca agcgcgccgg atcaggagct actaatttct
     2941 ctctgttaaa gcaggcagga gatgtggagg agaatccagg tcctcaattc accatgtcca
     3001 gactggacaa gagcaaagtc ataaacggcg ctctggaatt actcaatgga gtcggtatcg
     3061 aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct accctgtact
     3121 ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc aatcgagatg ctggacaggc
     3181 atcataccca cttctgcccc ctggaaggcg agtcatggca agactttctg cggaacaacg
     3241 ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg catctcggca
     3301 cccgcccaac agagaaacag tacgaaaccc tggaaaatca gctcgcgttc ctgtgtcagc
     3361 aaggcttctc cctggagaac gcactgtacg ctctgtccgc cgtgggccac tttacactgg
     3421 gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga ggaaagagag acacctacca
     3481 ccgattctat gcccccactt ctgagacaag caattgagct gttcgaccgg cagggagccg
     3541 aacctgcctt ccttttcggc ctggaactaa tcatatgtgg cctggagaaa cagctaaagt
     3601 gcgaaagcgg cgggccggcc gacgcccttg acgattttga cttagacatg ctcccagccg
     3661 atgcccttga cgactttgac cttgatatgc tgcctgctga cgctcttgac gattttgacc
     3721 ttgacatgct ccccgggtaa ctaagtaaga atccagacat gataagatac attgatgagt
     3781 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     3841 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     3901 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     3961 tctacaaatg tggtatggct gattatgatc ctgcaagcct cgtcgtcctg gccggaccac
     4021 gctatctgtg caaggtcccc ggccccggac gcgcgctcca tgagcagagc gcccgccgcc
     4081 gaggcgaaga ctcgggcggc gccctgcccg tcccaccagg tcaacaggcg gtaaccggcc
     4141 tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg gcggacggga
     4201 agtatccagc tcgaccaggc ttgtcgactc gaagaaccca gctttcttgt acaaagttgg
     4261 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     4321 taaaatcatt atttgccatc cagctgcagg gcggccgcga tatcccctat agtgagtcgt
     4381 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     4441 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     4501 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     4561 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     4621 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     4681 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     4741 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     4801 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     4861 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     4921 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     4981 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     5041 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     5101 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     5161 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     5221 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     5281 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     5341 attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac
     5401 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     5461 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5521 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5581 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5641 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5701 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5761 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5821 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5881 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5941 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6001 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6061 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6121 tgtt
//
