LOCUS       Exported                4023 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pMVP L3-L2 entry plasmid, contains eCFP-P2A + WPRE for 3- or 
            4-component MultiSite Gateway Pro assembly. Allows expression of 
            C-term eCFP linked by P2A to gene of interest..
ACCESSION   .
VERSION     .
KEYWORDS    LA201: pMVP (L3-L2) P2A-eCFP + WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4023)
  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 4023)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4023
                     /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             680..736
                     /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             743..1462
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(788..809)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1049..1068)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1396..1417
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1519..2107
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1572..1592)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1990..2001)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(2135..2234)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2264..2283)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2265..2283)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2288..2304)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2288..2304)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2406..2425)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2417..3226
                     /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(2484..2503)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3373..3961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3862..3881
                     /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 agttggtggc ggatccggag ctactaattt ctctctgtta aagcaggcag gagatgtgga
      721 ggagaatcca ggtcctaagg gaatggtgag caagggcgag gagctgttca ccggggtggt
      781 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
      841 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
      901 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcag
      961 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1021 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1081 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1141 ggacggcaac atcctggggc acaagctgga gtacaactac atcagccaca acgtctatat
     1201 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     1261 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1321 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1381 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1441 catggacgag ctgtacaagt aagaattctg aagcttagcc tagagctcct atagtgagac
     1501 cggttagtaa tgatcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     1561 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     1621 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     1681 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     1741 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     1801 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     1861 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     1921 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     1981 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     2041 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     2101 tccccgcctg gcgatggtac tgtcctagta cctgacccag ctttcttgta caaagttggc
     2161 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     2221 aaaatcatta tttgccatcc agctgcaggg cggccgcgat atcccctata gtgagtcgta
     2281 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     2341 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     2401 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     2461 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     2521 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     2581 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     2641 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     2701 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     2761 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     2821 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     2881 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     2941 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     3001 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     3061 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     3121 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     3181 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     3241 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     3301 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     3361 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3421 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3481 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3541 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3601 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3661 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3721 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3781 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3841 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3901 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3961 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4021 gtt
//
