LOCUS       sequence-292322-        5395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pX333 backbone expressing 2 sgRNA targeting BFP and KRAS G12D to 
            edit mouse KRAS G12D allele..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5395)
  AUTHORS   Budagyan K, Chernoff J
  TITLE     A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell 
            Background.
  JOURNAL   Methods Mol Biol. 2021;2262:323-334. doi: 
            10.1007/978-1-0716-1190-6_20.
  PUBMED    33977487
REFERENCE   2  (bases 1 to 5395)
  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: "Methods Mol
            Biol."; date: "2021"; volume: "2262:323-334. doi"; pages: " 
            10.1007/978-1-0716-1190-6_20"
COMMENT     Sequence Label: U6::sgBFP-U6::sgKrasG12D-pX333-red
FEATURES             Location/Qualifiers
     source          1..5395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..318
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        339..414
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        505..753
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        774..849
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        939..1224
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1226..1503
                     /label=chicken beta-actin promoter
     intron          1504..1731
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1743..2429
                     /codon_start=1
                     /product="far-red fluorescent variant of Heteractis crispa 
                     chromoprotein"
                     /label=HcRed1
                     /note="mammalian codon-optimized"
                     /translation="MVSGLLKESMRIKMYMEGTVNGHYFKCEGEGDGNPFAGTQSMRIH
                     VTEGAPLPFAFDILAPCCEYGSRTFVHHTAEIPDFFKQSFPEGFTWERTTTYEDGGILT
                     AHQDTSLEGNCLIYKVKVHGTNFPADGPVMKNKSGGWEPSTEVVYPENGVLCGRNVMAL
                     KVGDRHLICHHYTSYRSKKAVRALTMPGFHFTDIRLQMLRKKKDEYFELYEASVARYSD
                     LPEKAN"
     primer_bind     complement(2454..2471)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2460..2667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2676..2816
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2676..2805
                     /label=AAV2 ITR
     rep_origin      2891..3346
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2978..2997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3188..3209
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3363..3382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3482..3504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3542..3560)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3628..3732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3733..3801,3802..4593)
                     /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     complement(3951..3970)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4764..5352
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5253..5272
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tcacatgcgc cattctcgag catgcgccat tctcgagcat gctcgagaat
       61 ggcgcatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      121 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
      181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      301 tggaaaggac gaaacaccgc actgcacccc gtggctcagt tttagagcta gaaatagcaa
      361 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      421 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca
      481 attctgcaga caaatggctc tagagagggc ctatttccca tgattccttc atatttgcat
      541 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      601 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      661 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      721 gctttatata tcttgtggaa aggacgaaac accgtggttg gagctgatgg cgtgttttag
      781 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      841 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
      901 ttagcgcgtg cgccaattct gcagacaaat ggggtacccg ttacataact tacggtaaat
      961 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata
     1021 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1081 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1141 gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1201 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1261 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1321 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     1381 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     1441 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     1501 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     1561 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     1621 cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg
     1681 gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg
     1741 gtatggtgag cggcctgctg aaggagagta tgcgcatcaa gatgtacatg gagggcaccg
     1801 tgaacggcca ctacttcaag tgcgagggcg agggcgacgg caaccccttc gccggcaccc
     1861 agagcatgag aatccacgtg accgagggcg cccccctgcc cttcgccttc gacatcctgg
     1921 ccccctgctg cgagtacggc agcaggacct tcgtgcacca caccgccgag atccccgact
     1981 tcttcaagca gagcttcccc gagggcttca cctgggagag aaccaccacc tacgaggacg
     2041 gcggcatcct gaccgcccac caggacacca gcctggaggg caactgcctg atctacaagg
     2101 tgaaggtgca cggcaccaac ttccccgccg acggccccgt gatgaagaac aagagcggcg
     2161 gctgggagcc cagcaccgag gtggtgtacc ccgagaacgg cgtgctgtgc ggccggaacg
     2221 tgatggccct gaaggtgggc gaccggcacc tgatctgcca ccactacacc agctaccgga
     2281 gcaagaaggc cgtgcgcgcc ctgaccatgc ccggcttcca cttcaccgac atccggctcc
     2341 agatgctgcg gaagaagaag gacgagtact tcgagctgta cgaggccagc gtggcccggt
     2401 acagcgacct gcccgagaag gccaactgag aattcctaga gctcgctgat cagcctcgac
     2461 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2521 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2581 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2641 ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc
     2701 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2761 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     2821 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     2881 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2941 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     3001 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3061 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     3121 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3181 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     3241 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     3301 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     3361 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3421 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3481 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3541 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3601 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3661 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3721 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3781 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3841 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3901 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3961 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4021 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4081 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4141 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4201 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4261 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4321 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4381 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4441 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4501 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4561 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4621 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4681 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4741 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4801 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4861 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4921 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4981 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5041 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5101 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5161 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5221 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5281 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5341 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgct
//
