LOCUS       sequence-486153-        3970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone containing an mClover3 fused human KRAS4A
            G12D. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3970)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, 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 for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 3970)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pME-V5-mClover-KRAS4A-G12D (JDW 813)
FEATURES             Location/Qualifiers
     source          1..3970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(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    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      687..696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             696..737
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(753..755,756..758,759..1469)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(798..819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1406..1427
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2095..2194)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2211..2230)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2212..2230)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2235..2251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2235..2251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2353..2372)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2364..3173
                     /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(2431..2450)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3320..3908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3809..3828
                     /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 tgtacaaaaa
      661 agcaggctgg atccactagt gctagcgcca ccatgggtaa gcctatccct aaccctctcc
      721 tcggtctcga ttctacgggt ggtgctggtg gtatggtgag caagggcgag gagctgttca
      781 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
      841 tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca
      901 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg
      961 cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1021 ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc tacaagaccc
     1081 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1141 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccact
     1201 acgtctatat cacggccgac aagcagaaga actgcatcaa ggctaacttc aagatccgcc
     1261 acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1321 gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc aagctgagca
     1381 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1441 ttacacatgg catggacgag ctgtacaagt actcctccgg actcagatct ggaggctccg
     1501 gaggcatgac tgaatataaa cttgtggtag ttggagctga tggcgtaggc aagagtgcct
     1561 tgacgataca gctaattcag aatcattttg tggacgaata tgatccaaca atagaggatt
     1621 cctacaggaa gcaagtagta attgatggag aaacctgtct cttggatatt ctcgacacag
     1681 caggtcaaga ggagtacagt gcaatgaggg accagtacat gaggactggg gagggctttc
     1741 tttgtgtatt tgccataaat aatactaaat catttgaaga tattcaccat tatagagaac
     1801 aaattaaaag agttaaggac tctgaagatg tacctatggt cctagtagga aataaatgtg
     1861 atttgccttc tagaacagta gacacaaaac aggctcagga cttagcaaga agttatggaa
     1921 ttccttttat tgaaacatca gcaaagacaa gacagagagt ggaggatgct ttttatacat
     1981 tggtgaggga gatccgacaa tacagattga aaaaaatcag caaagaagaa aagactcctg
     2041 gctgtgtgaa aattaaaaaa tgcattataa tgtaaggcgc gccctcgaga gatctcccag
     2101 ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt tgcaacgaac
     2161 aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg
     2221 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     2281 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     2341 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
     2401 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     2461 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
     2521 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     2581 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     2641 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
     2701 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     2761 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     2821 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2881 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     2941 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     3001 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     3061 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     3121 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     3181 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     3241 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     3301 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3361 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3421 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3481 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3541 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3601 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3661 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3721 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3781 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3841 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3901 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3961 ctcacatgtt
//
