LOCUS       Exported                3361 bp ds-DNA     circular SYN 08-MAR-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3361)
  AUTHORS   Link Hyrule
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jul 22, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3361
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     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     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     550..568
                     /label=NheI-KRAS(WT)-F
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     645..697
                     /label=JDW 415
     primer_bind     668..782
                     /label=JDW1221-F-BamHI-SpeI-NheI-Kozak-V5-linker-mTagBFP2
     regulatory      687..696
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             696..737
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             738..752
                     /codon_start=1
                     /product="Gly Gly Ala Gly Gly Flexible Linker"
                     /label=G2AG2 Linker
                     /label=3X Flag GlyGlyAlaGlyGly
                     /note="Sabourin M et al., A flexible protein linker 
                     improves the function of epitope-tagged proteins in 
                     Saccharomyces cerevisiae (2007). Yeast."
                     /translation="GGAGG"
     CDS             753..1463
                     /codon_start=1
                     /label=mTagBFP2
                     /note="Subach et al PLoS One. 2011;6(12):e28674. Epub 2011 
                     Dec "
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     753..782
                     /label=JDW 1196
     CDS             771..1463
                     /codon_start=1
                     /label=mTagBFP (aa 3-223)
                     /translation="ELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVE
                     GGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQ
                     DTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLV
                     GGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDL
                     PSKLGHKLN"
     primer_bind     790..813
                     /label=F-mTagBFP2-1
     primer_bind     complement(877..898)
                     /label=REV-mTagBFP2-2
     primer_bind     1379..1401
                     /label=F-mTagBFP2-2
     primer_bind     complement(1419..1465)
                     /label=JDW 1233-REV-mTagBFP2-BglIILinker
     primer_bind     complement(1429..1486)
                     /label=R-mTagBFP2-Long
     primer_bind     complement(1429..1463)
                     /label=mTagBFP2-REV-p2A
     primer_bind     complement(1441..1463)
                     /label=REV-mTagBFP2-1
     protein_bind    complement(1486..1585)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1586..1606)
                     /label=PmeI-HindIII-KRAS(WT)-R
     promoter        complement(1603..1621)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1626..1642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1755..2564
                     /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"
     misc_feature    2695..2704
                     /label=TCF/LEF DBD
     rep_origin      2711..3299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 tgtacaaaaa
      661 agcaggctgg atccactagt gctagcgcca ccatgggtaa gcctatccct aaccctctcc
      721 tcggtctcga ttctacgggt ggtgctggtg gtatggtgtc taagggcgaa gagctgatta
      781 aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt
      841 gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg
      901 tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca
      961 gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg
     1021 agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc
     1081 aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact
     1141 tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga
     1201 cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg
     1261 gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa cccgctaaga
     1321 acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca
     1381 acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta
     1441 gcaaactggg gcacaagctt aattaaggcg cgccctcgag agatctccca gctttcttgt
     1501 acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta
     1561 tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt
     1621 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     1681 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     1741 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     1801 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1861 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     1921 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     1981 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     2041 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     2101 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     2161 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     2221 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2281 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2341 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     2401 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2461 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     2521 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     2581 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa
     2641 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     2701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2821 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3361 t
//
