LOCUS       sequence-484481-        3964 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Gateway middle entry clone containing an mTagBFP2 fused human KRAS4A
            WT. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3964)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, Mosser LE, Fujimoto E, Chien CB, Kwan KM, 
            Arenkiel BR, Devine WP, Wythe JD
  TITLE     Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 3964)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pME-V5-mTagBFP2-KRAS4A-WT (JDW 831)
FEATURES             Location/Qualifiers
     source          1..3964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      151..160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             160..201
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             217..927
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    complement(1553..1652)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1669..1688)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1670..1688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1693..1709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1693..1709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1811..1830)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1822..2631
                     /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(1889..1908)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2778..3366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3267..3286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3520..3537
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3696..3723)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3815..3901)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3950..3964,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3964,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctggatccac tagtgctagc gccaccatgg gtaagcctat ccctaaccct
      181 ctcctcggtc tcgattctac gggtggtgct ggtggtatgg tgtctaaggg cgaagagctg
      241 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
      301 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
      361 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
      421 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
      481 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
      541 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
      601 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
      661 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
      721 gtgggcggga gccatctgat cgcaaacgcc aagaccacat atagatccaa gaaacccgct
      781 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
      841 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
      901 cctagcaaac tggggcacaa gcttaattac tcctccggac tcagatctgg aggctccgga
      961 ggcatgactg aatataaact tgtggtagtt ggagctggtg gcgtaggcaa gagtgccttg
     1021 acgatacagc taattcagaa tcattttgtg gacgaatatg atccaacaat agaggattcc
     1081 tacaggaagc aagtagtaat tgatggagaa acctgtctct tggatattct cgacacagca
     1141 ggtcaagagg agtacagtgc aatgagggac cagtacatga ggactgggga gggctttctt
     1201 tgtgtatttg ccataaataa tactaaatca tttgaagata ttcaccatta tagagaacaa
     1261 attaaaagag ttaaggactc tgaagatgta cctatggtcc tagtaggaaa taaatgtgat
     1321 ttgccttcta gaacagtaga cacaaaacag gctcaggact tagcaagaag ttatggaatt
     1381 ccttttattg aaacatcagc aaagacaaga cagagagtgg aggatgcttt ttatacattg
     1441 gtgagggaga tccgacaata cagattgaaa aaaatcagca aagaagaaaa gactcctggc
     1501 tgtgtgaaaa ttaaaaaatg cattataatg taaggcgcgc cctcgagaga tctcccagct
     1561 ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag
     1621 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag
     1681 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     1741 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     1801 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     1861 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     1921 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     1981 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     2041 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     2101 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     2161 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     2221 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     2281 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     2341 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     2401 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     2461 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     2521 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     2581 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     2641 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     2701 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     2761 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2821 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2881 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     2941 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3001 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3061 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3121 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3181 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3241 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3301 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3361 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3421 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3481 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3541 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3601 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg
     3661 cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     3721 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     3781 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     3841 ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt
     3901 gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc ccagtcacga
     3961 cgtt
//
