LOCUS       sequence-424629-        8708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SlugCBE-NNG and puromycin resistance genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8708)
  AUTHORS   Qi T, Wang Y, Yang Y, Gao S, Liu J, Huang Q, Tian Y, Tang J, Zheng 
            WV, Wang Y
  TITLE     Phage-assisted evolution of compact Cas9 variants targeting a simple
            NNG PAM.
  JOURNAL   Nat Chem Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5.
  PUBMED    38052959
REFERENCE   2  (bases 1 to 8708)
  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: "Nat Chem 
            Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5."
COMMENT     Sequence Label: pCMV-SlugCBE-NNG-P2A-puro
FEATURES             Location/Qualifiers
     source          1..8708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        160..363
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     314..334
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     405..424
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        405..423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             473..493
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4463..4711
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4742..4990
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5033..5053
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5063..5119
                     /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             5120..5716
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5120..5139)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5616..5636
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5729..5749
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5758..5775
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5798..5815)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5804..6028
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6099..6115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6099..6115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6112..6134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6123..6139
                     /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(6147..6153,6154..6171,6172..6177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6192..6213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6323..6340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6494..7082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6574..6593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7253..8044,8045..8113))
                     /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     7876..7895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8114..8218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8297..8316)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8488..8708,1..159)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
       61 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      121 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      181 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      241 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      301 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      361 gctggtttag tgaaccgtca gatccgctag agatccgcgg ccgctaatac gactcactat
      421 agggagagcc gccaccatga aacggacagc cgacggaagc gagttcgagt caccaaagaa
      481 gaagcggaaa gtcagcagtg aaaccggacc agtggcagtg gacccaaccc tgaggagacg
      541 gattgagccc catgaatttg aagtgttctt tgacccaagg gagctgagga aggagacatg
      601 cctgctgtac gagatcaagt ggggcacaag ccacaagatc tggcgccaca gctccaagaa
      661 caccacaaag cacgtggaag tgaatttcat cgagaagttt acctccgagc ggcacttctg
      721 cccctctacc agctgttcca tcacatggtt tctgtcttgg agcccttgcg gcgagtgttc
      781 caaggccatc accgagttcc tgtctcagca ccctaacgtg accctggtca tctacgtggc
      841 ccggctgtat caccacatgg accagcagaa caggcagggc ctgcgcgatc tggtgaattc
      901 tggcgtgacc atccagatca tgacagcccc agagtacgac tattgctggc ggaacttcgt
      961 gaattatcca cctggcaagg aggcacactg gccaagatac ccacccctgt ggatgaagct
     1021 gtatgcactg gagctgcacg caggaatcct gggcctgcct ccatgtctga atatcctgcg
     1081 gagaaagcag ccccagctga catttttcac cattgctctg cagtcttgtc actatcagcg
     1141 gctgcctcct catattctgt gggctacagg cctgaagtcc ggaggatcta gcggaggctc
     1201 ctctggctct gagacacctg gcacaagcga gagcgcaaca cctgaaagca gcgggggcag
     1261 cagcgggggg tcaaaccaaa aattcatact gggactggcc atcggaatca ccagcgtggg
     1321 ctacggcctg atcgactacg agacaaagaa tatcatcgat gccggcgtta gactgttccc
     1381 cgaggccaac gtggaaaaca acgagggaag aaggtccaaa cgtggaagca gaagactgaa
     1441 gcgacgccgc attcacagac ttgaacgggt gaagaagctg ctcgaggatt ataatctgct
     1501 ggatcagtcc cagattcctc agtctacaaa cccctacgcc atccgcgtga agggcctgtc
     1561 tgaagccctg agcaaggacg aactcgtgat tgccctgctc catatcgcca agagaagagg
     1621 catccacaag atcgacgtga tcgacagcaa cgacgacgtg gggaacgagc tcagcaccaa
     1681 ggaacagctg aataagaaca gcaagctgct gaaagacaaa tttgtgtgcc agatccagct
     1741 ggaaagaatg aatgagggcc aggtgcgggg agagaaaaac cggttcaaga ccgctgatat
     1801 catcaaggaa atcatccagc tgctgaatgt gcagaagaac ttccaccagc tggacgagaa
     1861 cttcatcaac aagtacatcg aactggttga gatgagacgg gaatacttcg agggccccgg
     1921 caagggcagt ccatatggct gggaaggcga ccctaaggct tggtacgaga cactgatggg
     1981 ccactgcacc tacttcccag atgagctgag aagcgtgaaa tacgcctaca gtgccgacct
     2041 gttcaacgct ctgaacgacc tgaacaacct ggtcatccaa agagatggac tgtctaagct
     2101 cgagtatcat gagaagtatc acatcatcga gaacgtgttc aagcagaaga agaaacctac
     2161 actgaagcag atcgccaatg agatcaatgt caaccctgaa gatatcaagg gctacagaat
     2221 cacaaagtct ggcaagcccc agtttaccga gtttaagctc taccacgacc tgaaaagcgt
     2281 gctgtttgac cagagcatcc tggagaacga agacgtgctg gaccagatcg ctgagatcct
     2341 gaccatctac caggacaagg atagcatcaa atctaagctg acggaactgg acatcctgct
     2401 gaacgaggaa gataaggaaa acatcgccca gctgactggc tacaccggga cccaccggct
     2461 cagcctgaaa tgcatccggc tggtcctgga agagcagtgg tattctagcc ggaaccagat
     2521 ggaaatcttc acccacctga acattaagcc taagaagatc aacctgacag ccgccaacaa
     2581 gatcccgaag gctatgatcg acgagttcat cctgagccct gtggtgaaga ggaccttcgg
     2641 ccaggccatt aaccttatta acaagatcat agaaaagtac ggcgtgcctg aagatatcat
     2701 catcgagctg gccagagaaa ataatagcaa ggacaagcag aagttcatca atgagatgca
     2761 gaaaaagaac gagaacacca gaaagagaat taacgaaatc atcggcaagt atggcaacca
     2821 gaacgccaag agactggtcg agaagattag actgcacgac gagcaggagg gcaagtgcct
     2881 gtactcactg gaaagcatcc ctctggagga cctgctgaac aaccccaacc actacgaggt
     2941 ggaccacatc attccaagat ctgtgtcctt cgacaactct taccacaaca aagtgctcgt
     3001 gaagcagagc gagaactcca aaaaatccaa cctgacccct taccagtact ttaacagcgg
     3061 caagtccaag ctctcttaca accagtttaa acaacacatc ctgaacctga gcaagtccca
     3121 ggatagaatc agcaaaaaaa agaaagagta tctgctggaa gaacgggaca tcaacaagtt
     3181 cgaggtgcaa aaagagttca tcaatagaaa cctggtggat acccggtacg ccacaagaga
     3241 gctgacaaac tacctgaagg cctacttcag cgccaacaat atgaacgtga aggtgaaaac
     3301 gatcaacggc agcttcaccg attacctgcg gaaagtgtgg aagtttaaga aggaacggaa
     3361 ccacggctac aagcaccacg ccgaggacgc cctgattatc gctaatgccg atttcctgtt
     3421 caaagagaac aagaagctga aagccgtgaa ctctgtgctg gaaaaacctg agatcgagag
     3481 caagcagctg gatatccagg tggatagcga ggataactac agcgaaatgt tcatcatccc
     3541 taagcaggtc caggacatca aggacttcag aaacttcaag tacagccaca gagtggacaa
     3601 gaagcctaac agacggctga tcaacgatac actgtacagc acccggaaga aggacaactc
     3661 cacctacatc gtgcagacca tcaaagatat ctatgccaaa gataatacca ccctgaagaa
     3721 gcagtttgac aagtcacccg agaagttcct catgtaccaa cacgatccgc ggaccttcga
     3781 gaagttggaa gtgatcatga agcagtacgc taatgagaag aatcctctgg ccaagtacca
     3841 cgaggaaaca ggcgagtacc tgaccaaata cagcaaaaaa aacaacggcc ctatcgtgaa
     3901 aagcctgaag tacattggaa acaagctggg cagacaccta gatgtgaccc accagttcaa
     3961 gagcagcacc aagaagttgg tgaagcggag catcaagcct tatagattcg acgtctacct
     4021 gaccgacaag ggatataagt tcatcaccat cagctacctg gacgtgctga agaaagacaa
     4081 ttactactac atacccgaac agaagtacga caagctcaaa ctgggcaagg ccatcgacaa
     4141 aaacgccaag tttatcgcta gcttctacaa gaatgatctg atcaagctgg acggcgagat
     4201 ctacaagatc atcggcgtga gtagcgacac cagaaacatg atcgaactgg atctgcctga
     4261 catcagatac aaagaatact gcgagctgaa caatatcaag ggcaaaccta gaatcataaa
     4321 gaccatcggc aaaaaggtga atagcatcga aaaactgaca accgacgtgc tgggcaacgt
     4381 gttcaccaac acccagtaca caaaacctca gctgctgttc aagcgaggaa atagcggcgg
     4441 gagcggcggg agcgggggga gcactaatct gagcgacatc attgagaagg agactgggaa
     4501 acagctggtc attcaggagt ccatcctgat gctgcctgag gaggtggagg aagtgatcgg
     4561 caacaagcca gagtctgaca tcctggtgca caccgcctac gacgagtcca cagatgagaa
     4621 tgtgatgctg ctgacctctg acgcccccga gtataagcct tgggccctgg tcatccagga
     4681 ttctaacggc gagaataaga tcaagatgct gagcggagga tccggaggat ctggaggcag
     4741 caccaacctg tctgacatca tcgagaagga gacaggcaag cagctggtca tccaggagag
     4801 catcctgatg ctgcccgaag aagtcgaaga agtgatcgga aacaagcctg agagcgatat
     4861 cctggtccat accgcctacg acgagagtac cgacgaaaat gtgatgctgc tgacatccga
     4921 cgccccagag tataagccct gggctctggt catccaggat tccaacggag agaacaaaat
     4981 caaaatgctg tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa
     5041 gaagaggaaa gtcggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt
     5101 ggaggagaac cctggaccta tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     5161 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     5221 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     5281 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     5341 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     5401 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     5461 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca
     5521 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     5581 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     5641 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     5701 ccgcaagccc ggtgcctctg gtggttctcc caagaagaaa aggaaagtct aaccggtcat
     5761 catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     5821 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5881 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5941 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     6001 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     6061 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     6121 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6181 taggatgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6241 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc gggaagaggc
     6301 ggtttgcgta ttgggccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6361 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6421 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6481 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6541 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6601 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6661 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6721 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6781 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6841 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6901 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6961 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7021 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7081 aagaagatcc tttgatcttt tctacggggt ctgacactca gtggaacgaa aactcacgtt
     7141 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7201 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7261 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7321 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7381 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7441 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7501 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7561 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7621 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7681 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7741 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7801 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7861 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7921 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7981 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8041 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8101 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8161 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8221 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc
     8281 tagggtcgac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     8341 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     8401 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     8461 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
     8521 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     8581 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     8641 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     8701 ggtaaact
//
