LOCUS       Exported                5304 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMVP L3-L2 entry plasmid, contains polyA + EF1a-TETa-polyA for 3- or
            4-component MultiSite Gateway Pro assembly. Adds EF1a-driven TETa 
            downstream of gene of interest..
ACCESSION   .
VERSION     .
KEYWORDS    KA601: pMVP (L3-L2) pA; EF1a::TETa-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5304)
  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 5304)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5304
                     /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"
     polyA_signal    820..901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        919..2097
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1150..2088
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2045..2065
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2114..2132
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2174..2920
                     /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    2943..3064
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2954..2973)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3416..3515)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3546..3564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3569..3585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3569..3585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3687..3706)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3698..4507
                     /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(3765..3784)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4654..5242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5143..5162
                     /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 aacaaagctt cctagctcga tttaaataga taactgacta gaggatcata
      721 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
      781 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
      841 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      901 cctcgagtct agacgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      961 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     1021 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     1081 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1141 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     1201 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     1261 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     1321 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     1381 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     1441 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     1501 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     1561 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     1621 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     1681 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     1741 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     1801 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     1861 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     1921 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     1981 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     2041 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagga
     2101 attagcttgg tactaatacg actcactata gggagaccca agctggctag gtgaattcga
     2161 ctggcgcgcc accatgtcca gactggacaa gagcaaagtc ataaacggcg ctctggaatt
     2221 actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     2281 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc
     2341 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     2401 agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg
     2461 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     2521 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     2581 cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga
     2641 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     2701 gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     2761 cctggagaaa cagctaaagt gcgaaagcgg cgggccggcc gacgcccttg acgattttga
     2821 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga
     2881 cgctcttgac gattttgacc ttgacatgct ccccgggtaa ctaagtaaga atccagacat
     2941 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3001 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3061 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     3121 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc ctgcaagcct
     3181 cgtcgtcctg gccggaccac gctatctgtg caaggtcccc ggccccggac gcgcgctcca
     3241 tgagcagagc gcccgccgcc gaggcgaaga ctcgggcggc gccctgcccg tcccaccagg
     3301 tcaacaggcg gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca
     3361 tgtccccctg gcggacggga agtatccagc tcgaccaggc ttgtcgactc gaagaaccca
     3421 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     3481 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgcagg gcggccgcga
     3541 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     3601 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     3661 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     3721 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     3781 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     3841 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     3901 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     3961 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     4021 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     4081 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     4141 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     4201 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     4261 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     4321 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     4381 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     4441 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     4501 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     4561 ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     4621 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4681 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4741 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4801 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4861 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4921 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4981 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5041 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5101 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5161 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5221 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5281 ttgctggcct tttgctcaca tgtt
//
