LOCUS       Exported                5315 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMVP L3-L2 entry plasmid, contains HA tag-polyA + 
            RIP-eGFP-P2A-TETa-polyA for 3- or 4-component MultiSite Gateway Pro 
            assembly. Adds C-term HA tag to gene plus downstream RIP-driven 
            GFP-P2A-TETa.
ACCESSION   .
VERSION     .
KEYWORDS    KA701: pMVP (L3-L2) HA tag + pA; RIP::eGFP-P2A-TETa-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5315)
  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 5315)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5315
                     /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"
     CDS             1387..2103
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1387..2103
                     /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(1432..1453)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1693..1712)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2040..2061
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2119..2175
                     /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             2185..2931
                     /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    2954..3075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2965..2984)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3427..3526)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3556..3575)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3557..3575)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3580..3596)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3580..3596)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3698..3717)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3709..4518
                     /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(3776..3795)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4665..5253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5154..5173
                     /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 agtctagact gagcaagaat ccagctatca
      961 atagaaacta tgaaacagtt ccagggacaa agataccagg tccccaacaa ctgcaacttt
     1021 ctgggaaatg aggtggaaaa tgctcagcca aggaaaaaga gggccttacc ctctctggga
     1081 caatgattgt gctgtgaact gcttcatcag gccatctggc cccttgttaa taatctaatt
     1141 accctaggtc taagtagagt tgttgacgtc caatgagcgc tttctgcaga cttagcacta
     1201 ggcaagtgtt tggaaattac agcttcagcc cctctcgcca tctgcctacc tacccctcct
     1261 agagccctta atgggccaaa cggcaaagtc cagggggcag agaggaggtg ctttggacta
     1321 taaagctagt ggagacccag taactcccaa ccctaagtga ccagctacga attcgactgg
     1381 cgcgccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1441 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1501 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1561 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1621 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1681 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1741 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1801 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1861 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1921 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1981 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2041 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2101 aagcgcgccg gatcaggagc tactaatttc tctctgttaa agcaggcagg agatgtggag
     2161 gagaatccag gtcctcaatt caccatgtcc agactggaca agagcaaagt cataaacggc
     2221 gctctggaat tactcaatgg agtcggtatc gaaggcctga cgacaaggaa actcgctcaa
     2281 aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc
     2341 gatgccctgc caatcgagat gctggacagg catcataccc acttctgccc cctggaaggc
     2401 gagtcatggc aagactttct gcggaacaac gccaagtcat tccgctgtgc tctcctctca
     2461 catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc
     2521 ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac
     2581 gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa
     2641 gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact tctgagacaa
     2701 gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta
     2761 atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccggc cgacgccctt
     2821 gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg
     2881 ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccgggta actaagtaag
     2941 aatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     3001 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3061 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3121 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat
     3181 cctgcaagcc tcgtcgtcct ggccggacca cgctatctgt gcaaggtccc cggccccgga
     3241 cgcgcgctcc atgagcagag cgcccgccgc cgaggcgaag actcgggcgg cgccctgccc
     3301 gtcccaccag gtcaacaggc ggtaaccggc ctcttcatcg ggaatgcgcg cgaccttcag
     3361 catcgccggc atgtccccct ggcggacggg aagtatccag ctcgaccagg cttgtcgact
     3421 cgaagaaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     3481 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
     3541 ggcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     3601 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     3661 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     3721 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     3781 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     3841 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3901 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3961 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     4021 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     4081 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     4141 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     4201 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     4261 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     4321 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     4381 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     4441 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     4501 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     4561 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg
     4621 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4681 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4741 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4801 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4861 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4921 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4981 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5041 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5101 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5161 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5221 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5281 ttcctggcct tttgctggcc ttttgctcac atgtt
//
