LOCUS       sequence-424420-        7964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SlugABE-NNG and puromycin resistance genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7964)
  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 7964)
  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-SlugABE-NNG-P2A-puro
FEATURES             Location/Qualifiers
     source          1..7964
                     /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             4261..4281
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4291..4347
                     /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             4348..4944
                     /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(4348..4367)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4844..4864
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4957..4977
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4986..5003
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5026..5043)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5032..5256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5327..5343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5327..5343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5340..5362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5351..5367
                     /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(5375..5381,5382..5399,5400..5405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5420..5441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5551..5568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5722..6310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5802..5821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6481..7272,7273..7341))
                     /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     7104..7123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7342..7446)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7525..7544)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7716..7964,1..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 aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcaa accaaaaatt catactggga
     1081 ctggccatcg gaatcaccag cgtgggctac ggcctgatcg actacgagac aaagaatatc
     1141 atcgatgccg gcgttagact gttccccgag gccaacgtgg aaaacaacga gggaagaagg
     1201 tccaaacgtg gaagcagaag actgaagcga cgccgcattc acagacttga acgggtgaag
     1261 aagctgctcg aggattataa tctgctggat cagtcccaga ttcctcagtc tacaaacccc
     1321 tacgccatcc gcgtgaaggg cctgtctgaa gccctgagca aggacgaact cgtgattgcc
     1381 ctgctccata tcgccaagag aagaggcatc cacaagatcg acgtgatcga cagcaacgac
     1441 gacgtgggga acgagctcag caccaaggaa cagctgaata agaacagcaa gctgctgaaa
     1501 gacaaatttg tgtgccagat ccagctggaa agaatgaatg agggccaggt gcggggagag
     1561 aaaaaccggt tcaagaccgc tgatatcatc aaggaaatca tccagctgct gaatgtgcag
     1621 aagaacttcc accagctgga cgagaacttc atcaacaagt acatcgaact ggttgagatg
     1681 agacgggaat acttcgaggg ccccggcaag ggcagtccat atggctggga aggcgaccct
     1741 aaggcttggt acgagacact gatgggccac tgcacctact tcccagatga gctgagaagc
     1801 gtgaaatacg cctacagtgc cgacctgttc aacgctctga acgacctgaa caacctggtc
     1861 atccaaagag atggactgtc taagctcgag tatcatgaga agtatcacat catcgagaac
     1921 gtgttcaagc agaagaagaa acctacactg aagcagatcg ccaatgagat caatgtcaac
     1981 cctgaagata tcaagggcta cagaatcaca aagtctggca agccccagtt taccgagttt
     2041 aagctctacc acgacctgaa aagcgtgctg tttgaccaga gcatcctgga gaacgaagac
     2101 gtgctggacc agatcgctga gatcctgacc atctaccagg acaaggatag catcaaatct
     2161 aagctgacgg aactggacat cctgctgaac gaggaagata aggaaaacat cgcccagctg
     2221 actggctaca ccgggaccca ccggctcagc ctgaaatgca tccggctggt cctggaagag
     2281 cagtggtatt ctagccggaa ccagatggaa atcttcaccc acctgaacat taagcctaag
     2341 aagatcaacc tgacagccgc caacaagatc ccgaaggcta tgatcgacga gttcatcctg
     2401 agccctgtgg tgaagaggac cttcggccag gccattaacc ttattaacaa gatcatagaa
     2461 aagtacggcg tgcctgaaga tatcatcatc gagctggcca gagaaaataa tagcaaggac
     2521 aagcagaagt tcatcaatga gatgcagaaa aagaacgaga acaccagaaa gagaattaac
     2581 gaaatcatcg gcaagtatgg caaccagaac gccaagagac tggtcgagaa gattagactg
     2641 cacgacgagc aggagggcaa gtgcctgtac tcactggaaa gcatccctct ggaggacctg
     2701 ctgaacaacc ccaaccacta cgaggtggac cacatcattc caagatctgt gtccttcgac
     2761 aactcttacc acaacaaagt gctcgtgaag cagagcgaga actccaaaaa atccaacctg
     2821 accccttacc agtactttaa cagcggcaag tccaagctct cttacaacca gtttaaacaa
     2881 cacatcctga acctgagcaa gtcccaggat agaatcagca aaaaaaagaa agagtatctg
     2941 ctggaagaac gggacatcaa caagttcgag gtgcaaaaag agttcatcaa tagaaacctg
     3001 gtggataccc ggtacgccac aagagagctg acaaactacc tgaaggccta cttcagcgcc
     3061 aacaatatga acgtgaaggt gaaaacgatc aacggcagct tcaccgatta cctgcggaaa
     3121 gtgtggaagt ttaagaagga acggaaccac ggctacaagc accacgccga ggacgccctg
     3181 attatcgcta atgccgattt cctgttcaaa gagaacaaga agctgaaagc cgtgaactct
     3241 gtgctggaaa aacctgagat cgagagcaag cagctggata tccaggtgga tagcgaggat
     3301 aactacagcg aaatgttcat catccctaag caggtccagg acatcaagga cttcagaaac
     3361 ttcaagtaca gccacagagt ggacaagaag cctaacagac ggctgatcaa cgatacactg
     3421 tacagcaccc ggaagaagga caactccacc tacatcgtgc agaccatcaa agatatctat
     3481 gccaaagata ataccaccct gaagaagcag tttgacaagt cacccgagaa gttcctcatg
     3541 taccaacacg atccgcggac cttcgagaag ttggaagtga tcatgaagca gtacgctaat
     3601 gagaagaatc ctctggccaa gtaccacgag gaaacaggcg agtacctgac caaatacagc
     3661 aaaaaaaaca acggccctat cgtgaaaagc ctgaagtaca ttggaaacaa gctgggcaga
     3721 cacctagatg tgacccacca gttcaagagc agcaccaaga agttggtgaa gcggagcatc
     3781 aagccttata gattcgacgt ctacctgacc gacaagggat ataagttcat caccatcagc
     3841 tacctggacg tgctgaagaa agacaattac tactacatac ccgaacagaa gtacgacaag
     3901 ctcaaactgg gcaaggccat cgacaaaaac gccaagttta tcgctagctt ctacaagaat
     3961 gatctgatca agctggacgg cgagatctac aagatcatcg gcgtgagtag cgacaccaga
     4021 aacatgatcg aactggatct gcctgacatc agatacaaag aatactgcga gctgaacaat
     4081 atcaagggca aacctagaat cataaagacc atcggcaaaa aggtgaatag catcgaaaaa
     4141 ctgacaaccg acgtgctggg caacgtgttc accaacaccc agtacacaaa acctcagctg
     4201 ctgttcaagc gaggaaattc tggcggctca aaaagaaccg ccgacggcag cgaattcgag
     4261 cccaagaaga agaggaaagt cggaagcgga gctactaact tcagcctgct gaagcaggct
     4321 ggagacgtgg aggagaaccc tggacctatg accgagtaca agcccacggt gcgcctcgcc
     4381 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     4441 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     4501 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4561 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4621 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4681 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     4741 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     4801 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     4861 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     4921 tgcatgaccc gcaagcccgg tgcctctggt ggttctccca agaagaaaag gaaagtctaa
     4981 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5041 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5101 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5161 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5221 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5281 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5341 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5401 gtaaagccta ggatgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5461 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5521 gaagaggcgg tttgcgtatt gggccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5581 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5641 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5701 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5761 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5821 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5881 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5941 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6001 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6061 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6121 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6181 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6241 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6301 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacactcagt ggaacgaaaa
     6361 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6421 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6481 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6541 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6601 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6661 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6721 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6781 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6841 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6901 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6961 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7021 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7081 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7141 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7201 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7261 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7321 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7381 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7441 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc
     7501 cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7561 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     7621 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     7681 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     7741 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     7801 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     7861 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     7921 gtatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
