LOCUS       sequence-424418-        7370 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses SlugCas9-NNG and puromycin resistance genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7370)
  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 7370)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-SlugCas9-NNG-P2A-puro
FEATURES             Location/Qualifiers
     source          1..7370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3667..3687
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3697..3753
                     /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             3754..4350
                     /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(3754..3773)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4250..4270
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4363..4383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4392..4409
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4432..4449)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4438..4662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4733..4749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4733..4749)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4746..4768)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4757..4773
                     /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(4781..4787,4788..4805,4806..4811))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4826..4847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4957..4974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5128..5716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5208..5227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5887..6678,6679..6747))
                     /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     6510..6529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6748..6852)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6931..6950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7122..7370,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcaacca aaaattcata
      481 ctgggactgg acatcggaat caccagcgtg ggctacggcc tgatcgacta cgagacaaag
      541 aatatcatcg atgccggcgt tagactgttc cccgaggcca acgtggaaaa caacgaggga
      601 agaaggtcca aacgtggaag cagaagactg aagcgacgcc gcattcacag acttgaacgg
      661 gtgaagaagc tgctcgagga ttataatctg ctggatcagt cccagattcc tcagtctaca
      721 aacccctacg ccatccgcgt gaagggcctg tctgaagccc tgagcaagga cgaactcgtg
      781 attgccctgc tccatatcgc caagagaaga ggcatccaca agatcgacgt gatcgacagc
      841 aacgacgacg tggggaacga gctcagcacc aaggaacagc tgaataagaa cagcaagctg
      901 ctgaaagaca aatttgtgtg ccagatccag ctggaaagaa tgaatgaggg ccaggtgcgg
      961 ggagagaaaa accggttcaa gaccgctgat atcatcaagg aaatcatcca gctgctgaat
     1021 gtgcagaaga acttccacca gctggacgag aacttcatca acaagtacat cgaactggtt
     1081 gagatgagac gggaatactt cgagggcccc ggcaagggca gtccatatgg ctgggaaggc
     1141 gaccctaagg cttggtacga gacactgatg ggccactgca cctacttccc agatgagctg
     1201 agaagcgtga aatacgccta cagtgccgac ctgttcaacg ctctgaacga cctgaacaac
     1261 ctggtcatcc aaagagatgg actgtctaag ctcgagtatc atgagaagta tcacatcatc
     1321 gagaacgtgt tcaagcagaa gaagaaacct acactgaagc agatcgccaa tgagatcaat
     1381 gtcaaccctg aagatatcaa gggctacaga atcacaaagt ctggcaagcc ccagtttacc
     1441 gagtttaagc tctaccacga cctgaaaagc gtgctgtttg accagagcat cctggagaac
     1501 gaagacgtgc tggaccagat cgctgagatc ctgaccatct accaggacaa ggatagcatc
     1561 aaatctaagc tgacggaact ggacatcctg ctgaacgagg aagataagga aaacatcgcc
     1621 cagctgactg gctacaccgg gacccaccgg ctcagcctga aatgcatccg gctggtcctg
     1681 gaagagcagt ggtattctag ccggaaccag atggaaatct tcacccacct gaacattaag
     1741 cctaagaaga tcaacctgac agccgccaac aagatcccga aggctatgat cgacgagttc
     1801 atcctgagcc ctgtggtgaa gaggaccttc ggccaggcca ttaaccttat taacaagatc
     1861 atagaaaagt acggcgtgcc tgaagatatc atcatcgagc tggccagaga aaataatagc
     1921 aaggacaagc agaagttcat caatgagatg cagaaaaaga acgagaacac cagaaagaga
     1981 attaacgaaa tcatcggcaa gtatggcaac cagaacgcca agagactggt cgagaagatt
     2041 agactgcacg acgagcagga gggcaagtgc ctgtactcac tggaaagcat ccctctggag
     2101 gacctgctga acaaccccaa ccactacgag gtggaccaca tcattccaag atctgtgtcc
     2161 ttcgacaact cttaccacaa caaagtgctc gtgaagcaga gcgagaactc caaaaaatcc
     2221 aacctgaccc cttaccagta ctttaacagc ggcaagtcca agctctctta caaccagttt
     2281 aaacaacaca tcctgaacct gagcaagtcc caggatagaa tcagcaaaaa aaagaaagag
     2341 tatctgctgg aagaacggga catcaacaag ttcgaggtgc aaaaagagtt catcaataga
     2401 aacctggtgg atacccggta cgccacaaga gagctgacaa actacctgaa ggcctacttc
     2461 agcgccaaca atatgaacgt gaaggtgaaa acgatcaacg gcagcttcac cgattacctg
     2521 cggaaagtgt ggaagtttaa gaaggaacgg aaccacggct acaagcacca cgccgaggac
     2581 gccctgatta tcgctaatgc cgatttcctg ttcaaagaga acaagaagct gaaagccgtg
     2641 aactctgtgc tggaaaaacc tgagatcgag agcaagcagc tggatatcca ggtggatagc
     2701 gaggataact acagcgaaat gttcatcatc cctaagcagg tccaggacat caaggacttc
     2761 agaaacttca agtacagcca cagagtggac aagaagccta acagacggct gatcaacgat
     2821 acactgtaca gcacccggaa gaaggacaac tccacctaca tcgtgcagac catcaaagat
     2881 atctatgcca aagataatac caccctgaag aagcagtttg acaagtcacc cgagaagttc
     2941 ctcatgtacc aacacgatcc gcggaccttc gagaagttgg aagtgatcat gaagcagtac
     3001 gctaatgaga agaatcctct ggccaagtac cacgaggaaa caggcgagta cctgaccaaa
     3061 tacagcaaaa aaaacaacgg ccctatcgtg aaaagcctga agtacattgg aaacaagctg
     3121 ggcagacacc tagatgtgac ccaccagttc aagagcagca ccaagaagtt ggtgaagcgg
     3181 agcatcaagc cttatagatt cgacgtctac ctgaccgaca agggatataa gttcatcacc
     3241 atcagctacc tggacgtgct gaagaaagac aattactact acatacccga acagaagtac
     3301 gacaagctca aactgggcaa ggccatcgac aaaaacgcca agtttatcgc tagcttctac
     3361 aagaatgatc tgatcaagct ggacggcgag atctacaaga tcatcggcgt gagtagcgac
     3421 accagaaaca tgatcgaact ggatctgcct gacatcagat acaaagaata ctgcgagctg
     3481 aacaatatca agggcaaacc tagaatcata aagaccatcg gcaaaaaggt gaatagcatc
     3541 gaaaaactga caaccgacgt gctgggcaac gtgttcacca acacccagta cacaaaacct
     3601 cagctgctgt tcaagcgagg aaattctggc ggctcaaaaa gaaccgccga cggcagcgaa
     3661 ttcgagccca agaagaagag gaaagtcgga agcggagcta ctaacttcag cctgctgaag
     3721 caggctggag acgtggagga gaaccctgga cctatgaccg agtacaagcc cacggtgcgc
     3781 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     3841 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3901 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3961 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     4021 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     4081 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     4141 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     4201 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     4261 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     4321 acctggtgca tgacccgcaa gcccggtgcc tctggtggtt ctcccaagaa gaaaaggaaa
     4381 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     4441 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4501 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4561 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4621 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4681 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4741 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4801 taaagtgtaa agcctaggat gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4861 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4921 gcgcgggaag aggcggtttg cgtattgggc cgcttcctcg ctcactgact cgctgcgctc
     4981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5101 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5161 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5221 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5281 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5341 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5401 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5461 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5521 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5581 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5641 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5701 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa
     5761 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5821 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5881 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5941 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6001 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6061 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6121 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6181 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6241 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6301 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6361 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6421 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6481 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6541 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6601 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6661 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6721 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6781 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6841 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg
     6901 atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag
     6961 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7021 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7081 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     7141 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     7201 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7261 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     7321 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
//
