LOCUS       sequence-385761-        8708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AncBE4max and nSchCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8708)
  AUTHORS   Wang S, Mao H, Hou L, Hu Z, Wang Y, Qi T, Tao C, Yang Y, Zhang C, Li
            M, Liu H, Hu S, Chai R, Wang Y
  TITLE     Compact SchCas9 Recognizes the Simple NNGR PAM.
  JOURNAL   Adv Sci (Weinh). 2022 Feb;9(4):e2104789. doi: 
            10.1002/advs.202104789. Epub 2021 Dec 6.
  PUBMED    34874112
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: "Adv Sci 
            (Weinh)."; date: "2022-02"; volume: "9(4):e2104789. doi"; pages: " 
            10.1002/advs.202104789. Epub 2021 Dec 6"
COMMENT     Sequence Label: SchBE4max
FEATURES             Location/Qualifiers
     source          1..8708
                     /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             4435..4683
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4714..4962
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5005..5025
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5035..5091
                     /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             5092..5688
                     /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(5092..5111)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5588..5608
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5701..5721
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5730..5747
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5770..5787)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5776..6000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6071..6087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6071..6087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6084..6106)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6095..6111
                     /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(6119..6125,6126..6143,6144..6149))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6164..6185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6295..6312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6466..7054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6546..6565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7225..8016,8017..8085))
                     /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     7848..7867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8086..8190)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8269..8288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8460..8708,1..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 aagtcagcag tgaaaccgga
      481 ccagtggcag tggacccaac cctgaggaga cggattgagc cccatgaatt tgaagtgttc
      541 tttgacccaa gggagctgag gaaggagaca tgcctgctgt acgagatcaa gtggggcaca
      601 agccacaaga tctggcgcca cagctccaag aacaccacaa agcacgtgga agtgaatttc
      661 atcgagaagt ttacctccga gcggcacttc tgcccctcta ccagctgttc catcacatgg
      721 tttctgtctt ggagcccttg cggcgagtgt tccaaggcca tcaccgagtt cctgtctcag
      781 caccctaacg tgaccctggt catctacgtg gcccggctgt atcaccacat ggaccagcag
      841 aacaggcagg gcctgcgcga tctggtgaat tctggcgtga ccatccagat catgacagcc
      901 ccagagtacg actattgctg gcggaacttc gtgaattatc cacctggcaa ggaggcacac
      961 tggccaagat acccacccct gtggatgaag ctgtatgcac tggagctgca cgcaggaatc
     1021 ctgggcctgc ctccatgtct gaatatcctg cggagaaagc agccccagct gacatttttc
     1081 accattgctc tgcagtcttg tcactatcag cggctgcctc ctcatattct gtgggctaca
     1141 ggcctgaagt ccggaggatc tagcggaggc tcctctggct ctgagacacc tggcacaagc
     1201 gagagcgcaa cacctgaaag cagcgggggc agcagcgggg ggtcaaccaa ttacatcctg
     1261 ggcctggcca tcggcatcac aagcgtgggc tacggtatcg tgaactccga gactagagac
     1321 atcatcgacg ccggagttag actgttcccc gaggccaacg tgaacaacaa tgaagggaga
     1381 agaagcaaga gaggcgcccg gagactcaaa aggcggcgca tccacagact ggaacgggtg
     1441 aagcagctgc tggccgagta tgaactggtg gacagcagca gcatccccca cagcacaaac
     1501 ccttacgaga tcagagttag aggcctgaga gaagagctga gcaaggacga gctagtcatc
     1561 gctctgctcc atatcgctaa gcgccgggga atccataacg tgaacgtggt catagacgac
     1621 aacgaaagca acaacgagct gagcacgaag gatcagctga aaaaaaatgc taaagccctg
     1681 gaggataagt acgtgtgcga gctgcaactt gaaagattca agcaggaggg caaagtgcgg
     1741 ggcgagagaa acagattcaa gaccgaagat ttcgtgaagg aagccagaca gctgctgaac
     1801 acccagacac agttccaccc catcgaccag aatttcatca accagtacat ccatctgatc
     1861 gaaaccagaa gagaatactt cgagggcccc ggagaaggct ccccttacgg ctgggaggga
     1921 aatatcaaga agtggtacga gcagatgatg ggccactgta cctactttcc agacgagctt
     1981 cggagcgtga agtacgccta cagcgctgat ctgttcaacg ccctgaatga cctgaacaat
     2041 ctggtgatca ccagagacga ggacagcaag ctcaactacg gcgagaaatt tcagatcatc
     2101 gaaaacgtgt tcaaacagaa gaaaacacct aatctgaagc agatcgccaa ggaaatcggc
     2161 gtgaacgaga cagacatcaa gggctataga gtgaataaga gcgacaagcc ccagtttacc
     2221 tctttcaagc tgtaccacga tctgaaggaa atcttcgagc acaccgaata cctgaacgac
     2281 gcccagctga tggacaacat cgccgagatc atcaccatct accaggaccc agagagcatc
     2341 atctctgcac tgaacgagct gcctgagctg ctgaccgaga atgagaagca gcagatctcc
     2401 cagctgacag gatactctgg cacacacaga ctgagcctga agtgcatcaa cctgctgatc
     2461 gacgacctct gggagagcag cctgaaccag atggaactgt tcaccaagct aaacctgaag
     2521 cctaagaagg tggacctgag ccagcaaaac aagatcccaa caaacctggt cgaggaattt
     2581 attctgagcc ctgtggtgaa gcgggccttt atccaatcta tcgaggtggt caatgccgtg
     2641 atcgaaaaat acggcttgcc tgatgatatc atcatcgaac tggcccggga gaacaacagc
     2701 gatgatagaa gaaagtttct gaagaaactg caaaaacaga atgaggaaac ccgcaagcag
     2761 gttgagaagg ttctgaaaga gtacggcaat gacaacgcca agagaatggt gcagaaaatc
     2821 aagctgcacg acatgcagga gggcaaatgt ctgtacagtc tgaaggacat acccctggac
     2881 gacctgctga gaaaccccac acactacgaa gtggaccaca tcatccccag aagcgtggcc
     2941 ttcgacaact caatccacaa caaggtgctg gtgagagctg aagaaaacag taaaaagggc
     3001 aaccggaccc cttaccagta cctgaacagc tctgaaagct ctctgagcta caacgaattc
     3061 aagcagcaca tcctaaacct ctctaagacc aaagaccgga tctcgaaaaa gaaacgagag
     3121 tacctgctgg aagaaagaga tatcaacaaa tttgacgtgc agaaggaatt catcaaccgg
     3181 aacctggttg atacacggta cgccaccaga gagctgacca gcctgctgaa agcctatttt
     3241 tccgccaatc agctggacgt gaaggtgaaa accatcaacg gcagcttcac caactacctg
     3301 agaaaggtgt ggaagttcga caaggaccgg aataagggct acaagcacca cgccgaggac
     3361 gccctgatca ttgccaacgc cgacttcctg tttaagcacg acaagaagct gaagaacgtg
     3421 accaagatcc tggatgcccc tggccaggag gtggacagca gccggaccag cgtgcagagc
     3481 gaggacgagt tcaacgagat gttcgaaaac acacagaagg tcaccgccat cagaaacttc
     3541 gagatccaga agttcagcca ccgggtggat aagaagccta accggcagct gataaacgac
     3601 accctgtaca gcaccagaaa catcgagggc accgagtacg tggtgcagac cctgaagaac
     3661 atctactcta aggacaacaa agatctgaag aagctcatcg agaaggaccc cagcaagttc
     3721 ctgatgtacc aacacgaccc aaagaccttc gagaagatcc agatgatcat gaagcaatac
     3781 tctgatgaaa gcaatcctct ggctaaatat tacgaggaaa agcaagagtt catcacaaaa
     3841 tacgccaaga gaggcgacgg ccctcctatc aagagtctga agtatatcgg caactccgtg
     3901 ggcagccacc tgcagattac aaaaggcttc aagaacagca actacaagct ggtgaaactc
     3961 tccaagaaat ccttcagatt tgacgtgtac aagacatctc agggctataa gatggttcct
     4021 atttcttacc tggatgtgaa gaagaaggac aactactacg tgatcccttg cgagatctat
     4081 agagatctga tgaaaatgaa gaaagtgaac aaggaggacc tgttcatcgg atcattctac
     4141 tacaacgatc tgatcaaggt ggaagatgat ctgtacaagg tgatcggcgt gaaccacatc
     4201 gagacaaacc ggattgaggt ggacaagatc gatatcagat acaaggacta ctgcgccctg
     4261 aacggcatca agacccctag actgtacaaa accatcggaa aaaacaccaa gaatatcgag
     4321 aagtacacca ccgacatcct gggaaatctc tataaggcta caccgcctaa gaaacctcag
     4381 ctgattttca agcgtggaat ggaaagcggc gggagcggcg ggagcggggg gagcactaat
     4441 ctgagcgaca tcattgagaa ggagactggg aaacagctgg tcattcagga gtccatcctg
     4501 atgctgcctg aggaggtgga ggaagtgatc ggcaacaagc cagagtctga catcctggtg
     4561 cacaccgcct acgacgagtc cacagatgag aatgtgatgc tgctgacctc tgacgccccc
     4621 gagtataagc cttgggccct ggtcatccag gattctaacg gcgagaataa gatcaagatg
     4681 ctgagcggag gatccggagg atctggaggc agcaccaacc tgtctgacat catcgagaag
     4741 gagacaggca agcagctggt catccaggag agcatcctga tgctgcccga agaagtcgaa
     4801 gaagtgatcg gaaacaagcc tgagagcgat atcctggtcc ataccgccta cgacgagagt
     4861 accgacgaaa atgtgatgct gctgacatcc gacgccccag agtataagcc ctgggctctg
     4921 gtcatccagg attccaacgg agagaacaaa atcaaaatgc tgtctggcgg ctcaaaaaga
     4981 accgccgacg gcagcgaatt cgagcccaag aagaagagga aagtcggaag cggagctact
     5041 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tatgaccgag
     5101 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     5161 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     5221 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     5281 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     5341 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     5401 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     5461 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     5521 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     5581 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     5641 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctc tggtggttct
     5701 cccaagaaga aaaggaaagt ctaaccggtc atcatcacca tcaccattga gtttaaaccc
     5761 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5821 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5881 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5941 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     6001 cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg
     6061 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     6121 acatacgagc cggaagcata aagtgtaaag cctaggatgc ctaatgagtg agctaactca
     6181 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6241 attaatgaat cggccaacgc gcgggaagag gcggtttgcg tattgggccg cttcctcgct
     6301 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6361 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6421 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6481 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6541 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6601 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6661 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6721 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6781 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6841 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6901 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6961 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7021 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7081 gtctgacrct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7141 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7201 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7261 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7321 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7381 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7441 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7501 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7561 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7621 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7681 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7741 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7801 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7861 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7921 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7981 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8041 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8101 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8161 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     8221 cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc
     8281 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     8341 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8401 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     8461 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     8521 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     8581 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     8641 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     8701 agtgtatc
//
