LOCUS       sequence-356900-        7767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Neisseria lactamica Type I-C Cas3 with NLS and His tag on
            the C terminus for protein purification in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7767)
  AUTHORS   Tan R, Krueger RK, Gramelspacher MJ, Zhou X, Xiao Y, Ke A, Hou Z, 
            Zhang Y
  TITLE     Cas11 enables genome engineering in human cells with compact 
            CRISPR-Cas3 systems.
  JOURNAL   Mol Cell. 2022 Feb 17;82(4):852-867.e5. doi: 
            10.1016/j.molcel.2021.12.032. Epub 2022 Jan 19.
  PUBMED    35051351
REFERENCE   2  (bases 1 to 7767)
  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"; doi: 
            "10.1016/j.molcel.2021.12.032"; journalName: "Mol Cell"; date: 
            "2022-02-17- 17"; volume: "82"; issue: "4"; pages: "852-867.e5"
COMMENT     Sequence Label: pET28-T7-NlaCas3-NLS-6xHis
FEATURES             Location/Qualifiers
     source          1..7767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(164..184)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             2708..2730
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2745..2769
                     /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)."
     primer_bind     complement(2769..2788)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2770..2788)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2872..2891)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3097..3174
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3175..4257
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3194..3213)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5066..5257
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5251..5273)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5359..5498
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5414..5433
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5513..5530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5684..6272)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5764..5783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6383..6402)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6394..7209
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             6394..7209
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6467..6486)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7301..7756)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7438..7459)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7650..7669
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagcaccttc cgcttcttct
      181 tggggcccac agagcctcct aaaaccgaat tttcaggtag ccaagccgca tcgtcaaaat
      241 ccgcgccaag cacggctttg taatagccct tatctaacac gagcaatccc gctcttgaaa
      301 aaacggcatg ttcaggcaac tttttcagtt cgtgttcgta cacagtaatc gtgtagcgtt
      361 gcagcttgcg gtaaacccat ttgtgtttga gcgggtcgct ttccaacaga ccgaaccagc
      421 tttcaaacgg ctcgggcagc agtggttgat cggtttgccc aaaggagttg tctggcggct
      481 gcggaaagcg ttgtttgtcc aaaatatcct gccattctga aacttcaaca ccatctagat
      541 gtcgtctgaa aaaatcgtcc agctcgtttg cttcgacgat ttgcggactg ccgtcttttt
      601 cccaatgagc caacgggata aacggcacaa tgagtgccac gccttggtta tcaatcaggt
      661 ggaaacgttc ggcagctgtg cggaatttaa ttgccagcgg attttcattt gatgcttctg
      721 ccgtcaaaag cgttgtgata ccgtgtttgt ccacatcacc tttgccgttg aatcggcgga
      781 aatattcggc aaatgccaac ggggaaagcg ggtcatcaag cagccctgct ttcagcatct
      841 cttcggtaat gtcctgcccc tgtttcaggc tgccgctggg cgcgccttct tcggcgcgga
      901 atacgactac ttcgcccaac tgcggcagtt taccttcacg gttgcaccgt cccgccgcct
      961 gggcaatgct gtccagccct gccatcgccc gatacacgca agggaaatcc aaatccacgc
     1021 ctgcttcaat caactgcgtg ctgaccagcc acaagggctt gtgcaggctg cctgcgcgat
     1081 acagtgccaa atatcggcga accaacgcaa tcacttcgct gcagtgtgtg gcgcacatat
     1141 tggcagataa atgtagcttg attccgttag aaggcagggc ggcaaagagt ttttgggcgt
     1201 gttttcgcgt attgaccact gccaaaacac acgggcgcgc ggctatttca tcggcaattt
     1261 tctgccagct ttgcgtttcg ccgtttggcg gcggcatttt gatgcggacg cggcgcagtt
     1321 tttccgataa ggcaattttg tctgccacaa tttcgcgcac atctggtagc ccttccaaaa
     1381 tagtgcgacc gaatgcgtcg atatttttgc caagctccgg ttgggttgcc gtgcacagca
     1441 caaaggtaac gccgtaatca cgcgccagca cccgcatcat gtcggtaatc ggtttttgga
     1501 aatcgcgcgg aagctgctgg gcttcatcca aaatcaccac gctgtcggca atattgtgaa
     1561 tcttgcggca acggctggtt ttcgccgcaa acaggctttc aaacagttgc acattggtag
     1621 taacaatcag cggcgcgtcc caattttccg tagcaagacg agttttcgct gtttccttat
     1681 cttctttcac ttccaaattg ctgtggtgtt ctaaaaccac atcatcgcct aatgcattgc
     1741 ggaaaacatt ggcattctgc tcgataatac tggtgaaagg aatagcatag ataatacgtt
     1801 ttttgccaaa tttcagcgcg tgcttcaaag cgaagcccaa gctcgccaaa gttttaccgc
     1861 caccggtcgg cacggttaaa gaaaacaaag tacggtccgt ttctgcggca gaaaaacatt
     1921 gctgcaaaat ggcgtggcgt tcttggttta aagatgaatt tttatctgct ttttctgaaa
     1981 gttgcgccat atattgctca tatcgccggt gtaactcatc caaaccggga aattttgggc
     2041 gcaatccggc agcctgcgca gtatctgcat cggcatagcc gttcataaag gcttcggtat
     2101 ccaaaaaatc ggcatccacc aagcaggaaa agagaaaacg catccaaata tgcaattctt
     2161 ccagctttgc cccgtcttcc caaaacgcaa aaaaatcccg catcaagtca tcatctgata
     2221 acgggaaaaa atcttcgggc aaactacttt ccacaaagcc cgacaaagac gctgccaact
     2281 cgtcatccgc ctgttgcaga cggcgtttca ggctgccttt gtcataccaa tctgccagcc
     2341 cagcatgatg cccggcaatc aaatatgcca gcaaatgccc gaaaaatgga tttagacgtt
     2401 ctaccgcata tttggcaccg gcagtggaat gcggaatttt gcgcaacttg gtagattcga
     2461 catcttccaa atgggcattt tctttttcaa agccggatgc attacggata tatttctgaa
     2521 aagattcctg aaatttcccc aaatcgtgca aaagccccgc atattcggca aacaacgacc
     2581 cataacgccc tgcaaaacgc ttggcgagtt gggcgacttt ttgtagatgt ttttgcaggg
     2641 gatgggaatg ccaatttttt gatgagtctt ggcgagcgtg ggctatatag tcgaaattca
     2701 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     2761 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     2821 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     2881 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     2941 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     3001 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     3061 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     3121 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     3181 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     3241 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     3301 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     3361 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     3421 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     3481 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     3541 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     3601 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     3661 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     3721 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     3781 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     3841 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     3901 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     3961 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     4021 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     4081 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     4141 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4201 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4261 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     4321 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     4381 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     4441 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     4501 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     4561 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     4621 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     4681 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     4741 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     4801 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     4861 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     4921 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     4981 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     5041 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     5101 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     5161 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     5221 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5281 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5341 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     5401 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     5461 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     5521 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5581 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5641 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5701 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5761 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5821 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5881 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5941 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6001 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6061 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6121 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6181 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6241 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6301 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     6361 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     6421 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     6481 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     6541 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     6601 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     6661 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     6721 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     6781 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     6841 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     6901 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     6961 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     7021 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     7081 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     7141 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     7201 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     7321 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     7381 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     7441 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     7501 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     7561 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7621 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7681 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     7741 ccgctacagg gcgcgtccca ttcgcca
//
