LOCUS       sequence-275138-        6677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR SONIC:  Kras G12D luciferase donor plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  AUTHORS   Mou H, Ozata DM, Smith JL, Sheel A, Kwan SY, Hough S, Kucukural A, 
            Kennedy Z, Cao Y, Xue W
  TITLE     CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in 
            vivo cancer modeling.
  JOURNAL   Genome Med. 2019 Apr 16;11(1):21. doi: 10.1186/s13073-019-0627-9.
  PUBMED    30987660
REFERENCE   2  (bases 1 to 6677)
  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: "Genome 
            Med."; date: "2019-04-16"; volume: "11(1):21. doi"; pages: " 
            10.1186/s13073-019-0627-9"
COMMENT     Sequence Label: H516 SONIC KrasG12D-IRES-luciferase donor
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    813..1379
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    829..1381
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             1393..3045
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSVLLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPGGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1459..1477)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(3094..3111)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3100..3324
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3325..3358)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3404..3422)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3443..3459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3443..3459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3456..3478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3467..3483
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3491..3497,3498..3515,3516..3521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3536..3557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3674..3691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3845..4433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3925..3944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4604..5395,5396..5464))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5227..5246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5465..5569)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5595..6050)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5732..5753)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5944..5963
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6177..6199
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6191..6208
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6192..6208
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6215..6234
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6215..6233
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6266..6282
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6266..6282
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(6326..6677,1..216)
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    join(6343..6677,1..218)
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
ORIGIN
        1 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
       61 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
      121 tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
      181 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca tgactgagta
      241 taaacttgtg gtggttggag ctgatggcgt aggcaagagc gccttgacga tacagctaat
      301 tcagaatcac tttgtggatg agtacgaccc tacgatagaa gactcctaca ggaaacaagt
      361 agtaattgat ggagaaacct gtctcttgga tattctcgac acagcaggtc aagaggagta
      421 cagtgcaatg agggaccagt acatgagaac tggggagggc tttctttgtg tatttgccat
      481 aaataatact aaatcatttg aagatattca ccattataga gaacaaatta aaagagtaaa
      541 ggactctgaa gatgtgccta tggtcctggt agggaataag tgtgatttgc cttctagaac
      601 agtagacacg aaacaggctc aggagttagc aaggagttac gggattccgt tcattgagac
      661 ctcagcaaag acaagacagg gtgttgacga tgccttctat acattagtcc gagaaattcg
      721 aaaacataaa gaaaagatga gcaaagatgg gaagaagaag aagaagaagt caaggacaag
      781 gtgtacagtt atgtgagtcg aggttaacga attccgcccc cccccctaac gttactggcc
      841 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
      901 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
      961 ggggtctttc ccctctcgcc aaaggaatgc aaggcctgtt gaatgtcgtg aaggaagcag
     1021 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     1081 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     1141 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     1201 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     1261 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     1321 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     1381 atggccacaa ccatggaaga cgccaaaaac ataaagaaag gcccggcgcc attctatccg
     1441 ctggaagatg gaaccgctgg agagcaactg cataaggcta tgaagagata cgccctggtt
     1501 cctggaacaa ttgcttttac agatgcacat atcgaggtgg acatcactta cgctgagtac
     1561 ttcgaaatgt ccgttcggtt ggcagaagct atgaaacgat atgggctgaa tacaaatcac
     1621 agaatcgtcg tatgcagtga aaactctctt caattcttta tgccggtgtt gggcgcgtta
     1681 tttatcggag ttgcagttgc gcccgcgaac gacatttata atgaacgtga attgctcaac
     1741 agtatgggca tttcgcagcc taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt
     1801 ttgaacgtgc aaaaaaagct cccaatcatc caaaaaatta ttatcatgga ttctaaaacg
     1861 gattaccagg gatttcagtc gatgtacacg ttcgtcacat ctcatctacc tcccggtttt
     1921 aatgaatacg attttgtgcc agagtccttc gatagggaca agacaattgc actgatcatg
     1981 aactcctctg gatctactgg tctgcctaaa ggtgtcgctc tgcctcatag gactgcctgc
     2041 gtgagattct cgcatgccag agatcctatt tttggcaatc aaatcattcc ggatactgcg
     2101 attttaagtg ttgttccatt ccatcacggt tttggaatgt ttactacact cggatatttg
     2161 atatgtggat ttcgagtcgt cttaatgtat agatttgaag aagagctgtt tctgaggagc
     2221 cttcaggatt acaagattca aagtgtgctg ctggtgccaa ccctattctc cttcttcgcc
     2281 aaaagcactc tgattgacaa atacgattta tctaatttac acgaaattgc ttctggtggc
     2341 gctcccctct ctaaggaagt cggggaagcg gttgccaaga ggttccatct gccaggtatc
     2401 aggcaaggat atgggctcac tgagactaca tcagctattc tgattacacc cgggggggat
     2461 gataaaccgg gcgcggtcgg taaagttgtt ccattttttg aagcgaaggt tgtggatctg
     2521 gataccggga aaacgctggg cgttaatcaa agaggcgaac tgtgtgtgag aggtcctatg
     2581 attatgtccg gttatgtaaa caatccggaa gcgaccaacg ccttgattga caaggatgga
     2641 tggctacatt ctggagacat agcttactgg gacgaagacg aacacttctt catcgttgac
     2701 cgcctgaagt ctctgattaa gtacaaaggc tatcaggtgg ctcccgctga attggaatcc
     2761 atcttgctcc aacaccccaa catcttcgac gcaggtgtcg caggtcttcc cgacgatgac
     2821 gccggtgaac ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa
     2881 gagatcgtgg attacgtcgc cagtcaagta acaaccgcga aaaagttgcg cggaggagtt
     2941 gtgtttgtgg acgaagtacc gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga
     3001 gagatcctca taaaggccaa gaagggcgga aagatcgccg tgtaattcta ggatccgtcg
     3061 acatgcatcg atgatctaga gctcgctaat cagcctcgac tgtgccttct agttgccagc
     3121 tatctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3181 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3241 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3301 ctggggatgc ggtgggctct atggataact tcgtatagca tacattatac gaagttatcg
     3361 gaacccttaa tgtcgagggg gggcccggta ccagcttttg ttccctttag tgagggttaa
     3421 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3481 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3541 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3601 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3661 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3721 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3781 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3841 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3901 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3961 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4021 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4081 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4141 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4201 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4261 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4321 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4381 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4441 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4501 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4561 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4621 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4681 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4741 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4801 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4861 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4921 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4981 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5041 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5101 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5161 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5221 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5281 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5341 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5401 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5461 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5521 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5581 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5641 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5701 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5761 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5821 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5881 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5941 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     6001 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     6061 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     6121 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     6181 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt
     6241 ggagctccac cgcggtggcg gccgctctag aactagtgga tccgagatcg agtgccgcat
     6301 caccggcaat tgacgcgtgc tcctctccct cccccccccc taacgttact ggccgaagcc
     6361 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     6421 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     6481 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     6541 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     6601 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     6661 cggcacaacc ccagtgc
//
