LOCUS       sequence-351030-        7888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized wt PlmCasX, 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 7888)
  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 7888)
  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: pCAT077
FEATURES             Location/Qualifiers
     source          1..7888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1033..1318
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1320..1597
                     /label=chicken beta-actin promoter
     intron          1598..1826
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1838..1847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1850..1870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4814..4834
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4835..4882
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4892..4948
                     /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             4949..5545
                     /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     5442..5462
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5579..5596)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5585..5792
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5801..5941
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5801..5930
                     /label=AAV2 ITR
     rep_origin      6016..6471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6103..6122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6313..6334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6488..6507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6607..6629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6667..6685)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6753..6857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6858..6926,6927..7718)
                     /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(7076..7095)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      901 gcgcttttat ctcattactt tgagagccat caccagcgac tatgtcgtat gggtaaagcg
      961 cttatttatc ggagaaaccg ataaataaga agcatcaaag gggtcttcga gaagaccctt
     1021 ttttttggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1081 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1141 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1201 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1261 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1321 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1381 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1441 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     1501 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     1561 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga cgctgccttc
     1621 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1681 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     1741 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     1801 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggccc caaagaagaa
     1861 gcggaaggtc agcatgcaag agatcaagag aatcaacaag atcagaagga gactggtcaa
     1921 ggacagcaac acaaagaagg ccggcaagac aggccccatg aaaaccctgc tcgtcagagt
     1981 gatgacccct gacctgagag agcggctgga aaacctgaga aagaagcccg agaacatccc
     2041 tcagcctatc agcaacacca gcagggccaa cctgaacaag ctgctgaccg actacaccga
     2101 gatgaagaaa gccatcctgc acgtgtactg ggaagagttc cagaaagacc ccgtgggcct
     2161 gatgagcaga gttgctcagc ccgctcctaa gaacatcgac cagagaaagc tgatccccgt
     2221 gaaggacggc aacgagagac tgacctctag cggctttgcc tgcagccagt gttgccagcc
     2281 tctgtacgtg tacaagctgg aacaagtgaa cgacaagggc aagccccaca ccaactactt
     2341 cggcagatgc aacgtgtccg agcacgagag gctgatcctg ctgtctcctc acaagcccga
     2401 ggccaacgat gagctggtca catacagcct gggcaagttc ggacagagag ccctggactt
     2461 ctacagcatc cacgtgacca gggagagcaa tcaccctgtg aagcccctgg aacagatcgg
     2521 cggcaatagc tgtgcctctg gacctgtggg aaaagccctg agcgacgcct gtatgggagc
     2581 cgtggcatcc ttcctgacca agtaccagga catcatcctg gaacaccaga aagtgatcaa
     2641 gaagaacgag aaaagactgg ccaacctcaa ggatatcgcc agcgctaacg gcctggcctt
     2701 tcctaagatc accctgcctc cacagcctca caccaaagag ggcatcgagg cctacaacaa
     2761 cgtggtggcc cagatcgtga tttgggtcaa cctgaatctg tggcagaagc tgaagatcgg
     2821 cagggacgaa gccaagccac tgcagagact gaagggcttc cctagcttcc ctctggtgga
     2881 aagacaggcc aatgaagtgg attggtggga catggtctgc aacgtgaaga agctgatcaa
     2941 cgagaagaaa gaggatggca aggttttctg gcagaacctg gccggctaca agagacaaga
     3001 agccctgctg ccttacctga gcagcgaaga ggaccggaag aagggcaaga agttcgccag
     3061 ataccagttc ggcgacctgc tgctgcacct ggaaaagaag cacggcgagg actggggcaa
     3121 agtgtacgat gaggcctggg agagaatcga caagaaggtg gaaggcctga gcaagcacat
     3181 taagctggaa gaggaaagaa ggagcgagga cgcccaatct aaagccgctc tgaccgattg
     3241 gctgagagcc aaggccagct ttgtgatcga gggcctgaaa gaggccgaca aggacgagtt
     3301 ctgcagatgc gagctgaagc tgcagaagtg gtacggcgat ctgagaggca agcccttcgc
     3361 cattgaggcc gagaacagca tcctggacat cagcggcttc agcaagcagt acaactgcgc
     3421 cttcatttgg cagaaagacg gcgtcaagaa actgaacctg tacctgatca tcaattactt
     3481 caaaggcggc aagctgcggt tcaagaagat caaacccgag gccttcgagg ctaacagatt
     3541 ctacaccgtg atcaacaaaa agtccggcga gatcgtgccc atggaagtga acttcaactt
     3601 cgacgacccc aacctgatta tcctgcctct ggccttcggc aagagacagg gcagagagtt
     3661 catctggaac gatctgctga gcctggaaac cggctctctg aagctggcca atggcagagt
     3721 gatcgagaaa accctgtaca acaggagaac cagacaggac gagcctgctc tgtttgtggc
     3781 cctgaccttc gagagaagag aggtgctgga cagcagcaac atcaagccca tgaacctgat
     3841 cggcatcgac cggggcgaga atatccctgc tgtgatcgcc ctgacagacc ctgaaggatg
     3901 cccactgagc agattcaagg actccctggg caaccctaca cacatcctga gaatcggcga
     3961 gagctacaaa gagaagcaga ggacaatcca ggccgccaaa gaggtggaac agagaagagc
     4021 cggcggatac tctaggaagt acgccagcaa ggccaagaat ctggccgacg acatggtccg
     4081 aaacaccgcc agagatctgc tgtactacgc cgtgacacag gacgccatgc tgatcttcga
     4141 gaatctgagc agaggcttcg gccggcaggg caagagaacc tttatggccg agaggcagta
     4201 caccagaatg gaagattggc tcacagctaa actggcctac gagggactgc ccagcaagac
     4261 ctacctgtcc aaaacactgg cccagtatac ctccaagacc tgcagcaatt gcggcttcac
     4321 catcaccagc gccgactacg acagagtgct ggaaaagctc aagaaaaccg ccaccggctg
     4381 gatgaccacc atcaacggca aagagctgaa ggttgagggc cagatcacct actacaacag
     4441 gtacaagagg cagaacgtcg tgaaggatct gagcgtggaa ctggacagac tgagcgaaga
     4501 gagcgtgaac aacgacatca gcagctggac aaagggcaga tcaggcgagg ctctgagcct
     4561 gctgaagaag aggtttagcc acagacctgt gcaagagaag ttcgtgtgcc tgaactgcgg
     4621 cttcgagaca cacgccgatg aacaggctgc cctgaacatt gccagaagct ggctgttcct
     4681 gagaagccaa gagtacaaga agtaccagac caacaagacc accggcaaca ccgacaagag
     4741 ggcctttgtg gaaacctggc agagcttcta cagaaaaaag ctgaaagaag tctggaagcc
     4801 cgccgtgact agtccaaaaa agaagagaaa ggtagattac aaagatgacg atgacaaaga
     4861 ctacaaggat gatgatgata agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     4921 cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac
     4981 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     5041 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     5101 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     5161 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     5221 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     5281 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     5341 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     5401 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     5461 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     5521 catgacccgc aagcccggtg cctgaacgcg ttaagaattc ctagagctcg ctgatcagcc
     5581 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     5641 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     5701 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     5761 gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag
     5821 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     5881 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     5941 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     6001 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6061 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     6121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6181 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     6241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     6361 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6421 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     6481 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     6541 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     6601 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     6661 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     6721 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6781 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     6841 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6901 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6961 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     7021 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     7081 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     7141 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     7201 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     7261 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     7321 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     7381 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     7441 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7501 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     7561 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     7621 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     7681 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7741 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7801 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     7861 accccgtaga aaagatcaaa ggatcttc
//
