LOCUS       sequence-359731-        7836 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 7836)
  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 7836)
  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..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        120..405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        complement(539..787)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(597..616)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(767..787)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(849..1437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(929..948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1608..2399,2400..2468))
                     /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     2231..2250
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2469..2573)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2641..2659
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2697..2719)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2819..2838
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2855..3310)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2992..3013)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3204..3223
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3385..3525
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3396..3525
                     /label=AAV2 ITR
     polyA_signal    3534..3741
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3730..3747
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3781..4377)
                     /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     complement(3864..3884)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(4378..4434)
                     /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             complement(4444..4491)
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             complement(4492..4512)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7456..7476)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7479..7488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          7500..7727
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa atacaaaatt
       61 gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct cacctcgacc
      121 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
      181 atgtactggg cacaatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
      241 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
      301 cattgacgtc aatggaaagt ccctattggc gttactattg acgtcaatgg gcgggggtcg
      361 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgggtac caaaaaaaag
      421 ggtcttctcg aagacccctt tgatgcttct tatttatcgg tttctccgat aaataagcgc
      481 tttacccata cgacatagtc gctggtgatg gctctcaaag taatgagata aaagcgccgg
      541 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
      601 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
      661 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
      721 caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
      781 ggccctcaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1261 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     1321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1501 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1561 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     1621 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1681 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1741 ataccgcggc ttccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1801 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1861 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     1921 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     1981 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2041 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2101 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     2161 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     2221 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     2281 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     2341 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     2401 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2461 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2521 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2581 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     2641 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     2701 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     2761 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     2821 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta
     2881 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc
     2941 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg
     3001 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     3061 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     3121 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3181 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg
     3241 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     3301 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa
     3361 ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg cgctcgctcg ctcactgagg
     3421 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
     3481 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgctccccag
     3541 catgcctgct attctcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca
     3601 gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga
     3661 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
     3721 gctggcaact agaaggcaca gtcgaggctg atcagcgagc tctaggaatt cttaacgcgt
     3781 tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc
     3841 ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc
     3901 caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct
     3961 gcccagaccc ttgccctggt ggtcgggcga gactccgacg gtggccagga accacgcggg
     4021 ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga
     4081 accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct
     4141 ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat
     4201 gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg
     4261 gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg
     4321 tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcgg
     4381 tccaggattc tcttcgacat ctccggcttg tttcagcaga gagaagtttg ttgcgccgga
     4441 tcccttatca tcatcatcct tgtagtcttt gtcatcgtca tctttgtaat ctacctttct
     4501 cttctttttt ggactagtca cggcgggctt ccagacttct ttcagctttt ttctgtagaa
     4561 gctctgccag gtttccacaa aggccctctt gtcggtgttg ccggtggtct tgttggtctg
     4621 gtacttcttg tactcttggc ttctcaggaa cagccagctt ctggcaatgt tcagggcagc
     4681 ctgttcatcg gcgtgtgtct cgaagccgca gttcaggcac acgaacttct cttgcacagg
     4741 tctgtggcta aacctcttct tcagcaggct cagagcctcg cctgatctgc cctttgtcca
     4801 gctgctgatg tcgttgttca cgctctcttc gctcagtctg tccagttcca cgctcagatc
     4861 cttcacgacg ttctgcctct tgtacctgtt gtagtaggtg atctggccct caaccttcag
     4921 ctctttgccg ttgatggtgg tcatccagcc ggtggcggtt ttcttgagct tttccagcac
     4981 tctgtcgtag tcggcgctgg tgatggtgaa gccgcaattg ctgcaggtct tggaggtata
     5041 ctgggccagt gttttggaca ggtaggtctt gctgggcagt ccctcgtagg ccagtttagc
     5101 tgtgagccaa tcttccattc tggtgtactg cctctcggcc ataaaggttc tcttgccctg
     5161 ccggccgaag cctctgctca gattctcgaa gatcagcatg gcgtcctgtg tcacggcgta
     5221 gtacagcaga tctctggcgg tgtttcggac catgtcgtcg gccagattct tggccttgct
     5281 ggcgtacttc ctagagtatc cgccggctct tctctgttcc acctctttgg cggcctggat
     5341 tgtcctctgc ttctctttgt agctctcgcc gattctcagg atgtgtgtag ggttgcccag
     5401 ggagtccttg aatctgctca gtgggcatcc ttcagggtct gtcagggcga tcacagcagg
     5461 gatattctcg ccccggtcga tgccgatcag gttcatgggc ttgatgttgc tgctgtccag
     5521 cacctctctt ctctcgaagg tcagggccac aaacagagca ggctcgtcct gtctggttct
     5581 cctgttgtac agggttttct cgatcactct gccattggcc agcttcagag agccggtttc
     5641 caggctcagc agatcgttcc agatgaactc tctgccctgt ctcttgccga aggccagagg
     5701 caggataatc aggttggggt cgtcgaagtt gaagttcact tccatgggca cgatctcgcc
     5761 ggactttttg ttgatcacgg tgtagaatct gttagcctcg aaggcctcgg gtttgatctt
     5821 cttgaaccgc agcttgccgc ctttgaagta attgatgatc aggtacaggt tcagtttctt
     5881 gacgccgtct ttctgccaaa tgaaggcgca gttgtactgc ttgctgaagc cgctgatgtc
     5941 caggatgctg ttctcggcct caatggcgaa gggcttgcct ctcagatcgc cgtaccactt
     6001 ctgcagcttc agctcgcatc tgcagaactc gtccttgtcg gcctctttca ggccctcgat
     6061 cacaaagctg gccttggctc tcagccaatc ggtcagagcg gctttagatt gggcgtcctc
     6121 gctccttctt tcctcttcca gcttaatgtg cttgctcagg ccttccacct tcttgtcgat
     6181 tctctcccag gcctcatcgt acactttgcc ccagtcctcg ccgtgcttct tttccaggtg
     6241 cagcagcagg tcgccgaact ggtatctggc gaacttcttg cccttcttcc ggtcctcttc
     6301 gctgctcagg taaggcagca gggcttcttg tctcttgtag ccggccaggt tctgccagaa
     6361 aaccttgcca tcctctttct tctcgttgat cagcttcttc acgttgcaga ccatgtccca
     6421 ccaatccact tcattggcct gtctttccac cagagggaag ctagggaagc ccttcagtct
     6481 ctgcagtggc ttggcttcgt ccctgccgat cttcagcttc tgccacagat tcaggttgac
     6541 ccaaatcacg atctgggcca ccacgttgtt gtaggcctcg atgccctctt tggtgtgagg
     6601 ctgtggaggc agggtgatct taggaaaggc caggccgtta gcgctggcga tatccttgag
     6661 gttggccagt cttttctcgt tcttcttgat cactttctgg tgttccagga tgatgtcctg
     6721 gtacttggtc aggaaggatg ccacggctcc catacaggcg tcgctcaggg cttttcccac
     6781 aggtccagag gcacagctat tgccgccgat ctgttccagg ggcttcacag ggtgattgct
     6841 ctccctggtc acgtggatgc tgtagaagtc cagggctctc tgtccgaact tgcccaggct
     6901 gtatgtgacc agctcatcgt tggcctcggg cttgtgagga gacagcagga tcagcctctc
     6961 gtgctcggac acgttgcatc tgccgaagta gttggtgtgg ggcttgccct tgtcgttcac
     7021 ttgttccagc ttgtacacgt acagaggctg gcaacactgg ctgcaggcaa agccgctaga
     7081 ggtcagtctc tcgttgccgt ccttcacggg gatcagcttt ctctggtcga tgttcttagg
     7141 agcgggctga gcaactctgc tcatcaggcc cacggggtct ttctggaact cttcccagta
     7201 cacgtgcagg atggctttct tcatctcggt gtagtcggtc agcagcttgt tcaggttggc
     7261 cctgctggtg ttgctgatag gctgagggat gttctcgggc ttctttctca ggttttccag
     7321 ccgctctctc aggtcagggg tcatcactct gacgagcagg gttttcatgg ggcctgtctt
     7381 gccggccttc tttgtgttgc tgtccttgac cagtctcctt ctgatcttgt tgattctctt
     7441 gatctcttgc atgctgacct tccgcttctt ctttggggcc atggtggcac cggtccaacc
     7501 tgaaaaaaag tgatttcagg caggtgctcc aggtaattaa acattaatac cccaccaacc
     7561 aaccatccct taaaccctta cctcttgctc agctaattac agcccggagg agaagggccg
     7621 tcccgcccgc tcacctgtgg gagtaacgcg gtcagtcaga gccggggcgg gcggcgcgag
     7681 gcggcggcgg agcggggcac ggggcgaagg cagcgcgcag cgactcccgc ccgccgcgcg
     7741 cttcgctttt tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc
     7801 gcgccgctct gattggctgc cgccgcacct ctccgc
//
