LOCUS       sequence-364076-        7865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized wt DpbCasX, sgRNAv2 
            scaffold, and restriction sites to clone in new spacers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7865)
  AUTHORS   Tsuchida CA, Zhang S, Doost MS, Zhao Y, Wang J, O'Brien E, Fang H, 
            Li CP, Li D, Hai ZY, Chuck J, Brotzmann J, Vartoumian A, Burstein D,
            Chen XW, Nogales E, Doudna JA, Liu JG
  TITLE     Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome 
            editing activity.
  JOURNAL   Mol Cell. 2022 Mar 17;82(6):1199-1209.e6. doi: 
            10.1016/j.molcel.2022.02.002. Epub 2022 Feb 25.
  PUBMED    35219382
REFERENCE   2  (bases 1 to 7865)
  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"; doi: 
            "10.1016/j.molcel.2022.02.002"; journalName: "Mol Cell"; date: 
            "2022-03-17- 17"; volume: "82"; issue: "6"; pages: "1199-1209.e6"
COMMENT     Sequence Label: pCAT079
FEATURES             Location/Qualifiers
     source          1..7865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      37..625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     526..545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        687..935
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     687..707
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     858..877
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1069..1354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1591..1818
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1830..1839
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1842..1862
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4827..4847
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4848..4895
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4905..4961
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4962..5558
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5455..5475
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5592..5609)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5598..5805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5814..5954
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5814..5943
                     /label=AAV2 ITR
     rep_origin      6029..6484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6116..6135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6326..6347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6501..6520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6620..6642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6680..6698)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6766..6870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6871..6939,6940..7731)
                     /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(7089..7108)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
       61 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
      121 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
      181 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
      241 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
      301 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
      361 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
      421 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
      481 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      541 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      601 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      661 cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc
      721 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
      781 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      841 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      901 tggctttata tatcttgtgg aaaggacgaa acaccggcgc ttttatctca ttactttgag
      961 agccatcacc agcgactatg tcgtatgggt aaagcgctta tttatcggag aaaccgataa
     1021 ataagaagca tcaaaggggt cttcgagaag accctttttt ttggtacccg ttacataact
     1081 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt
     1141 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     1201 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     1261 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca
     1321 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
     1381 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
     1441 attttgtgca gcgatggggg cggggggggg gggggggcga ggcggagagg tgcggcggca
     1501 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
     1561 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
     1621 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     1681 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
     1741 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
     1801 gaaatcactt tttttcaggt tggaccggtg ccaccatggc cccaaagaag aagcggaagg
     1861 tcagcgaaaa gagaatcaac aagatcagga agaagctgag cgccgacaac gccaccaagc
     1921 ctgtgtctag gagtggcccc atgaaaaccc tgctcgtcag agtgatgacc gacgacctga
     1981 agaagagact ggaaaagagg aggaagaaac ccgaagtgat gccccaagtg atcagcaaca
     2041 acgccgccaa caacctgaga atgctgctgg acgactacac caagatgaag gaagccatcc
     2101 tgcaggtcta ctggcaagag ttcaaggacg accacgtggg cctgatgtgc aagtttgccc
     2161 agcctgccag caagaagatc gaccagaaca agctgaagcc cgagatggac gagaagggca
     2221 atctgaccac agccggcttt gcctgctctc agtgtggcca gcctctgttc gtgtacaagc
     2281 tggaacaggt gtccgagaaa ggcaaggcct acaccaacta cttcggcaga tgtaacgtgg
     2341 ccgagcacga gaagctgatt ctgctggccc agctgaaacc tgagaaggac tctgatgagg
     2401 ccgtgaccta cagcctgggc aagtttggac agagagccct ggacttctac agcatccacg
     2461 tgaccaaaga aagcacacac cccgtgaagc ccctggctca gatcgccggc aatagatacg
     2521 cctctggacc tgtgggcaaa gccctgtccg atgcctgcat gggaacaatc gccagcttcc
     2581 tgagcaagta ccaggacatc atcatcgagc accagaaggt ggtcaagggc aaccagaaga
     2641 gactggaaag cctgagggag ctggccggca aagagaacct ggaatacccc agcgtgaccc
     2701 tgcctcctca gcctcacaca aaagaaggcg tggacgccta caacgaagtg atcgccagag
     2761 tgagaatgtg ggtcaacctg aacctgtggc agaagctgaa actgtccagg gacgacgcca
     2821 agcctctgct gagactgaag ggcttcccta gcttccccgt ggtggaaaga agggagaatg
     2881 aggtggactg gtggaacacc atcaacgaag tcaagaaact gatcgatgcc aagagagaca
     2941 tgggcagagt gttttggagc ggagtgaccg ccgagaagag aaacaccatc ctggaaggct
     3001 acaactacct gcctaacgag aacgaccaca agaagagaga gggcagcctg gaaaacccta
     3061 agaagcccgc caagagacag ttcggcgacc tgctgctgta cctggagaag aagtatgccg
     3121 gcgactgggg caaagtgttc gatgaggcct gggagagaat cgacaagaag attgccggcc
     3181 tgaccagcca catcgagaga gaggaagcca gaaacgccga ggacgcccag tctaaagccg
     3241 tgctgacaga ttggctgaga gccaaggcca gcttcgtgct ggaaaggctg aaagaaatgg
     3301 atgagaaaga gttctacgcc tgcgagatcc agctgcagaa gtggtacggg gacctgagag
     3361 gcaatccctt tgccgtggaa gccgagaaca gagtggtgga catcagcggc tttagcatcg
     3421 gctctgacgg ccactccatc cagtacagga acctgctggc ctggaagtac ctggaaaatg
     3481 gcaagagaga gttctacctg ctgatgaact acggcaagaa gggcagaatc agattcaccg
     3541 acggcaccga catcaagaaa agcggcaagt ggcagggcct gctgtatggc ggcggaaagg
     3601 ccaaagtgat cgacctgacc ttcgatcccg acgacgagca gctgatcatt ctgcctctgg
     3661 ccttcggcac cagacagggc agagagttca tctggaacga tctgctgtcc ctggaaaccg
     3721 gcctgatcaa gctggccaac ggcagggtga tcgagaaaac catctacaac aagaaaatcg
     3781 gcagagatga gcccgcactg tttgtggccc tgacctttga gaggagagaa gtggtcgatc
     3841 ccagcaacat caagcccgtg aacctgatcg gagtggacag aggcgagaat atccctgccg
     3901 tgattgccct gacagaccct gagggatgtc ccctgcctga gtttaaggat agctctggcg
     3961 gccctaccga catcctgaga atcggcgagg gctacaaaga gaagcagaga gccatccagg
     4021 ccgccaaaga ggtggaacag agaagagccg gcggatacag cagaaagttc gccagcaagt
     4081 ccagaaacct ggccgatgac atggtcagga acagcgccag ggacctgttc taccacgccg
     4141 tgacacatga tgccgtgctg gtgttcgaga acctgtcaag aggcttcggc agacagggaa
     4201 aaagaacctt catgaccgag agacagtaca cgaagatgga agattggctc accgccaaac
     4261 tggcctacga gggcctgaca agcaagacct acctgtccaa gacactggcc cagtacacct
     4321 ccaagacctg ctccaattgc ggcttcacca tcaccaccgc cgactacgat ggaatgctcg
     4381 tgagactgaa gaaaacctcc gacggctggg ccaccacact gaacaacaaa gagctgaaag
     4441 ccgagggcca gatcacctac tacaacagat acaagaggca gaccgtcgag aaagagctgt
     4501 ccgccgaact cgacagactg agcgaggaat ccggcaacaa cgacatcagc aagtggacca
     4561 agggacgcag agatgaggcc ctgttcctgc tgaagaagag gttcagccac agacctgtgc
     4621 aagagcagtt tgtgtgcctg gattgcggcc atgaagtgca cgcagatgaa caggccgctc
     4681 tgaatatcgc cagaagctgg ctgttcctga acagcaacag caccgagttc aagagctaca
     4741 agagcggcaa gcagcctttc gttggagcct ggcaggcctt ctacaagaga aggctcaaag
     4801 aagtgtggaa gcccaacgcc actagtccaa aaaagaagag aaaggtagat tacaaagatg
     4861 acgatgacaa agactacaag gatgatgatg ataagggatc cggcgcaaca aacttctctc
     4921 tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg
     4981 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     5041 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     5101 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5161 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     5221 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     5281 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     5341 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     5401 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     5461 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     5521 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagaa ttcctagagc
     5581 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5641 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5701 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5761 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     5821 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     5881 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     5941 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6001 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     6061 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     6121 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     6181 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     6241 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     6301 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     6361 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     6421 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     6481 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6541 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6601 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6661 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6721 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6781 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6841 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6901 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6961 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     7021 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     7081 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     7141 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     7201 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     7261 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     7321 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     7381 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     7441 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     7501 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7561 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     7621 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7681 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7741 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7801 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7861 cactg
//
