LOCUS       Exported                3733 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMVP L5-L4 entry plasmid, contains Hygro-P2A for 4-component 
            MultiSite Gateway Pro assembly. Allows expression of N-term Hygro 
            selection marker linked by P2A to gene of interest..
ACCESSION   .
VERSION     .
KEYWORDS    KO801: pMVP (L5-L4) Hygro-P2A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3733)
  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 3733)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3733
                     /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=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     5'UTR           680..722
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             734..1768
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             1750..1758
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1781..1837
                     /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"
     protein_bind    complement(1848..1943)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1974..1993)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1975..1993)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1998..2014)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1998..2014)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2116..2135)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2127..2936
                     /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(2194..2213)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3083..3671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3572..3591
                     /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 tgtatacaaa
      661 agttgtagaa tacaagcttc ttgttctttt tgcagaagct cagaataaac gctcaacttt
      721 ggcagatctg aacatgaaga agcccgaact caccgctacc agcgttgaaa aatttctcat
      781 cgagaagttc gacagtgtga gcgacctgat gcagttgtcg gagggcgaag agagccgagc
      841 cttcagcttc gatgtcggcg gacgcggcta tgtactgcgg gtgaatagct gcgctgatgg
      901 cttctacaaa gaccgctacg tgtaccgcca cttcgccagc gctgcactac ccatccccga
      961 agtgttggac atcggcgagt tcagcgagag cctgacatac tgcatcagta gacgcgccca
     1021 aggcgttact ctccaagacc tccccgaaac agagctgcct gctgtgttac agcctgtcgc
     1081 cgaagctatg gatgctattg ccgccgccga cctcagtcaa accagcggct tcggcccatt
     1141 cgggccccaa ggcatcggcc agtacacaac ctggcgggat ttcatttgcg ccattgctga
     1201 tccccatgtc taccactggc agaccgtgat ggacgacacc gtgtccgcca gcgtagctca
     1261 agccctggac gaactgatgc tgtgggccga agactgtccc gaggtgcgcc acctcgtcca
     1321 tgccgacttc ggcagcaaca acgtcctgac cgacaacggc cgcatcaccg ccgtaatcga
     1381 ctggtccgaa gctatgttcg gggacagtca gtacgaggtg gccaacatct tcttctggcg
     1441 gccctggctg gcttgcatgg agcagcagac tcgctacttc gagcgccggc atcccgagct
     1501 ggccggcagc cctcgtctgc gagcctacat gctgcgcatc ggcctggatc agctctacca
     1561 gagcctcgtg gacggcaact tcgacgatgc tgcctgggct caaggccgct gcgatgccat
     1621 cgtccgcagc ggggccggca ccgtcggtcg cacacaaatc gctcgccgga gcgcagccgt
     1681 atggaccgac ggctgcgtcg aggtgctggc cgacagcggc aaccgccggc ccagtacacg
     1741 accgcgcgct aaggaggtag gtcgagttgc cggatccgga gctactaatt tctctctgtt
     1801 aaagcaggca ggagatgtgg aggagaatcc aggtcctaag gcccacccaa cttttctata
     1861 caaagttggc attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat
     1921 cagtcaaaat aaaatcatta tttgccatcc agctgcaggg cggccgcgat atcccctata
     1981 gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc
     2041 tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt
     2101 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc
     2161 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     2221 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     2281 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     2341 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     2401 catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc
     2461 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     2521 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     2581 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     2641 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     2701 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     2761 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     2821 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     2881 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     2941 aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa
     3001 gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg
     3061 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3241 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3361 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3481 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3661 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3721 ttgctcacat gtt
//
