LOCUS       sequence-328010-        8685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ABEmax and nSlugCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8685)
  AUTHORS   Hu Z, Zhang C, Wang S, Gao S, Wei J, Li M, Hou L, Mao H, Wei Y, Qi 
            T, Liu H, Liu D, Lan F, Lu D, Wang H, Li J, Wang Y
  TITLE     Discovery and engineering of small SlugCas9 with broad targeting 
            range and high specificity and activity.
  JOURNAL   Nucleic Acids Res. 2021 Mar 15. pii: 6171296. doi: 
            10.1093/nar/gkab148.
  PUBMED    33721016
REFERENCE   2  (bases 1 to 8685)
  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: "Nucleic 
            Acids Res. 2021 Mar 15. pii: 6171296. doi: 10.1093/nar/gkab148."
COMMENT     Sequence Label: SlugABEmax
FEATURES             Location/Qualifiers
     source          1..8685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        200..403
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     354..374
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     445..464
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        445..463
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             513..533
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4923..4943
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4953..5009
                     /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             join(5010..5012,5013..5015,5016..5726)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5010..5012,5013..5015,5016..5726)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5055..5076)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5316..5335)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5663..5684
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5739..5759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5768..5785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5808..5825)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5814..6038
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6109..6125)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6109..6125)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6122..6144)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6133..6149
                     /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(6157..6163,6164..6181,6182..6187))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6202..6223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6340..6357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6511..7099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6591..6610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7270..8061,8062..8130))
                     /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     7893..7912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8131..8235)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8314..8333)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8505..8685,1..199)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
       61 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      121 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      181 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      241 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      301 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      361 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      421 gatccgctag agatccgcgg ccgctaatac gactcactat agggagagcc gccaccatga
      481 aacggacagc cgacggaagc gagttcgagt caccaaagaa gaagcggaaa gtctctgaag
      541 tcgagtttag ccacgagtat tggatgaggc acgcactgac cctggcaaag cgagcatggg
      601 atgaaagaga agtccccgtg ggcgccgtgc tggtgcacaa caatagagtg atcggagagg
      661 gatggaacag gccaatcggc cgccacgacc ctaccgcaca cgcagagatc atggcactga
      721 ggcagggagg cctggtcatg cagaattacc gcctgatcga tgccaccctg tatgtgacac
      781 tggagccatg cgtgatgtgc gcaggagcaa tgatccacag caggatcgga agagtggtgt
      841 tcggagcacg ggacgccaag accggcgcag caggctccct gatggatgtg ctgcaccacc
      901 ccggcatgaa ccaccgggtg gagatcacag agggaatcct ggcagacgag tgcgccgccc
      961 tgctgagcga tttctttaga atgcggagac aggagatcaa ggcccagaag aaggcacaga
     1021 gctccaccga ctctggagga tctagcggag gatcctctgg aagcgagaca ccaggcacaa
     1081 gcgagtccgc cacaccagag agctccggcg gctcctccgg aggatcctct gaggtggagt
     1141 tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca cgcgatgaga
     1201 gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc gagggctgga
     1261 acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc ctgagacagg
     1321 gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg acattcgagc
     1381 cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg gtgtttggcg
     1441 tgaggaacgc aaaaaccggc gccgcaggct ccctgatgga cgtgctgcac taccccggca
     1501 tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc gccctgctgt
     1561 gctatttctt tcggatgcct agacaggtgt tcaatgctca gaagaaggcc cagagctcca
     1621 ccgactccgg aggatctagc ggaggctcct ctggctctga gacacctggc acaagcgaga
     1681 gcgcaacacc tgaaagcagc gggggcagca gcggggggtc aaaccaaaaa ttcatactgg
     1741 gactggccat cggaatcacc agcgtgggct acggcctgat cgactacgag acaaagaata
     1801 tcatcgatgc cggcgttaga ctgttccccg aggccaacgt ggaaaacaac gagggaagaa
     1861 ggtccaaacg tggaagcaga agactgaagc gacgccgcat tcacagactt gaacgggtga
     1921 agaagctgct cgaggattat aatctgctgg atcagtccca gattcctcag tctacaaacc
     1981 cctacgccat ccgcgtgaag ggcctgtctg aagccctgag caaggacgaa ctcgtgattg
     2041 ccctgctcca tatcgccaag agaagaggca tccacaagat cgacgtgatc gacagcaacg
     2101 acgacgtggg gaacgagctc agcaccaagg aacagctgaa taagaacagc aagctgctga
     2161 aagacaaatt tgtgtgccag atccagctgg aaagaatgaa tgagggccag gtgcggggag
     2221 agaaaaaccg gttcaagacc gctgatatca tcaaggaaat catccagctg ctgaatgtgc
     2281 agaagaactt ccaccagctg gacgagaact tcatcaacaa gtacatcgaa ctggttgaga
     2341 tgagacggga atacttcgag ggccccggca agggcagtcc atatggctgg gaaggcgacc
     2401 ctaaggcttg gtacgagaca ctgatgggcc actgcaccta cttcccagat gagctgagaa
     2461 gcgtgaaata cgcctacagt gccgacctgt tcaacgctct gaacgacctg aacaacctgg
     2521 tcatccaaag agatggactg tctaagctcg agtatcatga gaagtatcac atcatcgaga
     2581 acgtgttcaa gcagaagaag aaacctacac tgaagcagat cgccaatgag atcaatgtca
     2641 accctgaaga tatcaagggc tacagaatca caaagtctgg caagccccag tttaccgagt
     2701 ttaagctcta ccacgacctg aaaagcgtgc tgtttgacca gagcatcctg gagaacgaag
     2761 acgtgctgga ccagatcgct gagatcctga ccatctacca ggacaaggat agcatcaaat
     2821 ctaagctgac ggaactggac atcctgctga acgaggaaga taaggaaaac atcgcccagc
     2881 tgactggcta caccgggacc caccggctca gcctgaaatg catccggctg gtcctggaag
     2941 agcagtggta ttctagccgg aatcagatgg aaatcttcac acacctgaac attaagccta
     3001 agaagatcaa cctgacagcc gccaacaaga tcccgaaggc tatgatcgac gagttcatcc
     3061 tgagccctgt ggtgaagagg accttcggcc aggccattaa ccttattaac aagatcatag
     3121 aaaagtacgg cgtgcctgaa gatatcatca tcgagctggc cagagaaaat aatagcaagg
     3181 acaagcagaa gttcatcaat gagatgcaga aaaagaacga gaacaccaga aagagaatta
     3241 acgaaatcat cggcaagtat ggcaaccaga acgccaagag actggtcgag aagattagac
     3301 tgcacgacga gcaggagggc aagtgcctgt actcactgga aagcatccct ctggaggacc
     3361 tgctgaacaa ccccaaccac tacgaggtgg accacatcat tccaagatct gtgtccttcg
     3421 acaactctta ccacaacaaa gtgctcgtga agcagagcga gaactccaaa aaatccaacc
     3481 tgacccctta ccagtacttt aacagcggca agtccaagct ctcttacaac cagtttaaac
     3541 aacacatcct gaacctgagc aagtcccagg atagaatcag caaaaaaaag aaagagtatc
     3601 tgctggaaga acgggacatc aacaagttcg aggtgcaaaa agagttcatc aatagaaacc
     3661 tggtggatac ccggtacgcc acaagagagc tgacaaacta cctgaaggcc tacttcagcg
     3721 ccaacaatat gaacgtgaag gtgaaaacga tcaacggcag cttcaccgat tacctgcgga
     3781 aagtgtggaa gtttaagaag gaacggaacc acggctacaa gcaccacgcc gaggacgccc
     3841 tgattatcgc taatgccgat ttcctgttca aagagaacaa gaagctgaaa gccgtgaact
     3901 ctgtgctgga aaaacctgag atcgagagca agcagctgga tatccaggtg gatagcgagg
     3961 ataactacag cgaaatgttc atcatcccta agcaggtcca ggacatcaag gacttcagaa
     4021 acttcaagta cagccacaga gtggacaaga agcctaacag acagctgatc aacgatacac
     4081 tgtacagcac ccggaagaag gacaactcca cctacatcgt gcagaccatc aaagatatct
     4141 atgccaaaga taataccacc ctgaagaagc agtttgacaa gtcacccgag aagttcctca
     4201 tgtaccaaca cgatccgcgg accttcgaga agttggaagt gatcatgaag cagtacgcta
     4261 atgagaagaa tcctctggcc aagtaccacg aggaaacagg cgagtacctg accaaataca
     4321 gcaaaaaaaa caacggccct atcgtgaaaa gcctgaagta cattggaaac aagctgggca
     4381 gccacctaga tgtgacccac cagttcaaga gcagcaccaa gaagttggtg aagctgagca
     4441 tcaagcctta tagattcgac gtctacctga ccgacaaggg atataagttc atcaccatca
     4501 gctacctgga cgtgctgaag aaagacaatt actactacat acccgaacag aagtacgaca
     4561 agctcaaact gggcaaggcc atcgacaaaa acgccaagtt tatcgctagc ttctacaaga
     4621 atgatctgat caagctggac ggcgagatct acaagatcat cggcgtgaat agcgacacca
     4681 gaaacatgat cgaactggat ctgcctgaca tcagatacaa agaatactgc gagctgaaca
     4741 atatcaaggg cgaacctaga atcaaaaaga ccatcggcaa aaaggtgaat agcatcgaaa
     4801 aactgacaac cgacgtgctg ggcaacgtgt tcaccaacac ccagtacaca aaacctcagc
     4861 tgctgttcaa gcgaggaaat tctggcggct caaaaagaac cgccgacggc agcgaattcg
     4921 agcccaagaa gaagaggaaa gtcggaagcg gagctactaa cttcagcctg ctgaagcagg
     4981 ctggagacgt ggaggagaac cctggaccta tggtgagcaa gggcgaggag ctgttcaccg
     5041 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     5101 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     5161 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctat ggagtgcagt
     5221 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     5281 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     5341 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     5401 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     5461 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     5521 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     5581 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     5641 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     5701 ctctcggcat ggacgagctg tacaagtctg gtggttctcc caagaagaag aggaaagtct
     5761 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     5821 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5881 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5941 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     6001 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     6061 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     6121 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6181 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6241 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6361 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6421 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6481 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6541 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6601 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6661 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6721 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6781 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6841 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6901 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6961 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7021 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7081 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg
     7141 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7201 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7261 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7321 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7381 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7441 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7501 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7561 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7621 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7681 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7741 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7801 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7861 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7921 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7981 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8041 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8101 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8221 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     8281 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     8341 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     8401 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8461 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     8521 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     8581 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     8641 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggac
//
