LOCUS       sequence-328011-        8028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AncBE4max and nSlugCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8028)
  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 8028)
  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: SlugBE4max
FEATURES             Location/Qualifiers
     source          1..8028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     246..265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        246..264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             314..334
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4313..4561
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4592..4840
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4883..4903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4912..4929
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4952..4969)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4958..5182
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5253..5269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5253..5269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5277..5293
                     /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(5301..5307,5308..5325,5326..5331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5346..5367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5735..5754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6414..7205,7206..7274))
                     /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     7037..7056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7275..7379)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7458..7477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7649..8028
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      241 gccgctaata cgactcacta tagggagagc cgccaccatg aaacggacag ccgacggaag
      301 cgagttcgag tcaccaaaga agaagcggaa agtcagcagt gaaaccggac cagtggcagt
      361 ggacccaacc ctgaggagac ggattgagcc ccatgaattt gaagtgttct ttgacccaag
      421 ggagctgagg aaggagacat gcctgctgta cgagatcaag tggggcacaa gccacaagat
      481 ctggcgccac agctccaaga acaccacaaa gcacgtggaa gtgaatttca tcgagaagtt
      541 tacctccgag cggcacttct gcccctctac cagctgttcc atcacatggt ttctgtcttg
      601 gagcccttgc ggcgagtgtt ccaaggccat caccgagttc ctgtctcagc accctaacgt
      661 gaccctggtc atctacgtgg cccggctgta tcaccacatg gaccagcaga acaggcaggg
      721 cctgcgcgat ctggtgaatt ctggcgtgac catccagatc atgacagccc cagagtacga
      781 ctattgctgg cggaacttcg tgaattatcc acctggcaag gaggcacact ggccaagata
      841 cccacccctg tggatgaagc tgtatgcact ggagctgcac gcaggaatcc tgggcctgcc
      901 tccatgtctg aatatcctgc ggagaaagca gccccagctg acatttttca ccattgctct
      961 gcagtcttgt cactatcagc ggctgcctcc tcatattctg tgggctacag gcctgaagtc
     1021 tggaggatct agcggaggat cctctggcag cgagacacca ggaacaagcg agtcagcaac
     1081 accagagagc agtggcggca gcagcggcgg cagcaaccaa aaattcatac tgggactggc
     1141 catcggaatc accagcgtgg gctacggcct gatcgactac gagacaaaga atatcatcga
     1201 tgccggcgtt agactgttcc ccgaggccaa cgtggaaaac aacgagggaa gaaggtccaa
     1261 acgtggaagc agaagactga agcgacgccg cattcacaga cttgaacggg tgaagaagct
     1321 gctcgaggat tataatctgc tggatcagtc ccagattcct cagtctacaa acccctacgc
     1381 catccgcgtg aagggcctgt ctgaagccct gagcaaggac gaactcgtga ttgccctgct
     1441 ccatatcgcc aagagaagag gcatccacaa gatcgacgtg atcgacagca acgacgacgt
     1501 ggggaacgag ctcagcacca aggaacagct gaataagaac agcaagctgc tgaaagacaa
     1561 atttgtgtgc cagatccagc tggaaagaat gaatgagggc caggtgcggg gagagaaaaa
     1621 ccggttcaag accgctgata tcatcaagga aatcatccag ctgctgaatg tgcagaagaa
     1681 cttccaccag ctggacgaga acttcatcaa caagtacatc gaactggttg agatgagacg
     1741 ggaatacttc gagggccccg gcaagggcag tccatatggc tgggaaggcg accctaaggc
     1801 ttggtacgag acactgatgg gccactgcac ctacttccca gatgagctga gaagcgtgaa
     1861 atacgcctac agtgccgacc tgttcaacgc tctgaacgac ctgaacaacc tggtcatcca
     1921 aagagatgga ctgtctaagc tcgagtatca tgagaagtat cacatcatcg agaacgtgtt
     1981 caagcagaag aagaaaccta cactgaagca gatcgccaat gagatcaatg tcaaccctga
     2041 agatatcaag ggctacagaa tcacaaagtc tggcaagccc cagtttaccg agtttaagct
     2101 ctaccacgac ctgaaaagcg tgctgtttga ccagagcatc ctggagaacg aagacgtgct
     2161 ggaccagatc gctgagatcc tgaccatcta ccaggacaag gatagcatca aatctaagct
     2221 gacggaactg gacatcctgc tgaacgagga agataaggaa aacatcgccc agctgactgg
     2281 ctacaccggg acccaccggc tcagcctgaa atgcatccgg ctggtcctgg aagagcagtg
     2341 gtattctagc cggaatcaga tggaaatctt cacacacctg aacattaagc ctaagaagat
     2401 caacctgaca gccgccaaca agatcccgaa ggctatgatc gacgagttca tcctgagccc
     2461 tgtggtgaag aggaccttcg gccaggccat taaccttatt aacaagatca tagaaaagta
     2521 cggcgtgcct gaagatatca tcatcgagct ggccagagaa aataatagca aggacaagca
     2581 gaagttcatc aatgagatgc agaaaaagaa cgagaacacc agaaagagaa ttaacgaaat
     2641 catcggcaag tatggcaacc agaacgccaa gagactggtc gagaagatta gactgcacga
     2701 cgagcaggag ggcaagtgcc tgtactcact ggaaagcatc cctctggagg acctgctgaa
     2761 caaccccaac cactacgagg tggaccacat cattccaaga tctgtgtcct tcgacaactc
     2821 ttaccacaac aaagtgctcg tgaagcagag cgagaactcc aaaaaatcca acctgacccc
     2881 ttaccagtac tttaacagcg gcaagtccaa gctctcttac aaccagttta aacaacacat
     2941 cctgaacctg agcaagtccc aggatagaat cagcaaaaaa aagaaagagt atctgctgga
     3001 agaacgggac atcaacaagt tcgaggtgca aaaagagttc atcaatagaa acctggtgga
     3061 tacccggtac gccacaagag agctgacaaa ctacctgaag gcctacttca gcgccaacaa
     3121 tatgaacgtg aaggtgaaaa cgatcaacgg cagcttcacc gattacctgc ggaaagtgtg
     3181 gaagtttaag aaggaacgga accacggcta caagcaccac gccgaggacg ccctgattat
     3241 cgctaatgcc gatttcctgt tcaaagagaa caagaagctg aaagccgtga actctgtgct
     3301 ggaaaaacct gagatcgaga gcaagcagct ggatatccag gtggatagcg aggataacta
     3361 cagcgaaatg ttcatcatcc ctaagcaggt ccaggacatc aaggacttca gaaacttcaa
     3421 gtacagccac agagtggaca agaagcctaa cagacagctg atcaacgata cactgtacag
     3481 cacccggaag aaggacaact ccacctacat cgtgcagacc atcaaagata tctatgccaa
     3541 agataatacc accctgaaga agcagtttga caagtcaccc gagaagttcc tcatgtacca
     3601 acacgatccg cggaccttcg agaagttgga agtgatcatg aagcagtacg ctaatgagaa
     3661 gaatcctctg gccaagtacc acgaggaaac aggcgagtac ctgaccaaat acagcaaaaa
     3721 aaacaacggc cctatcgtga aaagcctgaa gtacattgga aacaagctgg gcagccacct
     3781 agatgtgacc caccagttca agagcagcac caagaagttg gtgaagctga gcatcaagcc
     3841 ttatagattc gacgtctacc tgaccgacaa gggatataag ttcatcacca tcagctacct
     3901 ggacgtgctg aagaaagaca attactacta catacccgaa cagaagtacg acaagctcaa
     3961 actgggcaag gccatcgaca aaaacgccaa gtttatcgct agcttctaca agaatgatct
     4021 gatcaagctg gacggcgaga tctacaagat catcggcgtg aatagcgaca ccagaaacat
     4081 gatcgaactg gatctgcctg acatcagata caaagaatac tgcgagctga acaatatcaa
     4141 gggcgaacct agaatcaaaa agaccatcgg caaaaaggtg aatagcatcg aaaaactgac
     4201 aaccgacgtg ctgggcaacg tgttcaccaa cacccagtac acaaaacctc agctgctgtt
     4261 caagcgagga aatagcggcg ggagcggcgg gagcggcggg agcgggggga gcactaatct
     4321 gagcgacatc attgagaagg agactgggaa acagctggtc attcaggagt ccatcctgat
     4381 gctgcctgag gaggtggagg aagtgatcgg caacaagcca gagtctgaca tcctggtgca
     4441 caccgcctac gacgagtcca cagatgagaa tgtgatgctg ctgacctctg acgcccccga
     4501 gtataagcct tgggccctgg tcatccagga ttctaacggc gagaataaga tcaagatgct
     4561 gagcggagga tccggaggat ctggaggcag caccaacctg tctgacatca tcgagaagga
     4621 gacaggcaag cagctggtca tccaggagag catcctgatg ctgcccgaag aagtcgaaga
     4681 agtgatcgga aacaagcctg agagcgatat cctggtccat accgcctacg acgagagtac
     4741 cgacgaaaat gtgatgctgc tgacatccga cgccccagag tataagccct gggctctggt
     4801 catccaggat tccaacggag agaacaaaat caaaatgctg tctggcggct caaaaagaac
     4861 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaccgg tcatcatcac
     4921 catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     4981 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5041 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     5101 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     5161 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgataccg
     5221 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5281 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctaggat
     5341 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcgggaag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6481 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7381 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc
     7441 ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     7501 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     7561 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     7621 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     7681 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     7741 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     7801 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     7861 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     7921 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     7981 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatg
//
