pET-NlaCascade-NLS-6xHis

LOCUS       pET-NlaCascade-NLS-6xHis      8526 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expressing Neisseria lactamica Type I-C Cascade subunits 
            (Cas5-Cas8-Cas11-Cas7) with NLS and His tag on the C terminus of 
            Cas7 for protein purification in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8526)
  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 8526)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.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: pET-NlaCascade-NLS-6xHis
FEATURES             Location/Qualifiers
     source          1..8526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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)."
     RBS             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3393..3413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3420..3437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3490..3508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3504..3551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3588..4043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3675..3694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3885..3906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4135..4950)
                     /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             complement(4135..4950)
                     /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     4858..4877
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4942..4961
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5072..5660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5561..5580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5814..5831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5846..5985
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5911..5930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6071..6093
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6087..6278)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7087..8169)
                     /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
                     YIPPLTTIKQDFRLLGQTSVGRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8131..8150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8170..8247)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8453..8472
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      121 aggttcatcc tggaaatcag tggtgatttg gcatgcttca caaggtctga gctaaaggtg
      181 gaaagggtta gttatcctgt gataacgccg tctgccgcca ggaacatcct aatggcgata
      241 ttgtggaagc cggcgattcg ctggaaggtc ttgaagatag aaatcctaaa accgattcag
      301 tggacgaata tccgccgcaa cgaagtggga actaagatga gtgagcgtag cggctcgctc
      361 tatattgaag ataaccgcca gcagcgcgca tccatgctgc tgaaagacgt tgcctaccgc
      421 attcacgccg attttgacat gaccagtgaa gcgggcgaga gcgacaacta tgttaaattt
      481 gccgaaatgt tcaagcggcg ggcaaagaaa ggacaatatt tccaccaacc ttatttaggc
      541 tgtcgtgagt ttccttgtga tttcaggttg ctggaaaaag ccgaagatgg attgccactc
      601 gaagacatta cccaagattt cggttttatg ctgtatgaca tggatttcag caaatccgac
      661 ccgcgtgatt ccaataacgc cgagccgatg ttttaccaat gcaaagcggt aaacggcgtg
      721 attaccgtgc cgcctgccga cagcgaggag gtgaaacgat gattttgcac gcgctcaccc
      781 aatactatca acgcaaagcc gaaagtgatg gcggtattgc ccaggaaggg tttgaaaaca
      841 aagaaatacc gttcattatc gttatagaca aacagggtaa ttttattcag ctggaagata
      901 cccgtgagct gaaagttaag aagaaagttg gccgcacttt tttagtaccg aaaggtttgg
      961 gcaggagcgg ttcaaaatcc tacgaagtaa gcaatttatt gtgggatcac tacggttatg
     1021 tacttgctta tgccggagaa aaagggcagg agcaggcgga caaacagcat gccagcttta
     1081 ccgccaaagt aaatgaattg aaacaggcgc tgcccgatga tgcaggtgtt actgcggttg
     1141 ctgccttttt gtcttctgcg gaagaaaaaa gcaaagtcat gcaggctgca aattgggcgg
     1201 agtgtgccaa agtcaaaggc tgtaatctca gcttccgcct ggtggatgaa gcggtagatt
     1261 tggtttgcca gtcaaaggcg gtgcgggaat atgtgagtca agcaaatcaa acgcaatccg
     1321 ataatgtcca aaaaggcatt tgcctggtaa cgggcaaagc tgcgccgatt gcgcggctgc
     1381 ataacgccgt gaaaggcgtg aatgccaagc ccgccccgtt tgcatcggta aatctgtcgg
     1441 cttttgaatc atacggcaaa gagcagggct ttatctttcc cgtgggcgag caagccatgt
     1501 tcgaatatac caccgccttg aacaccttgc ttgctagcga aaaccgattc cgtatcggcg
     1561 atgtaacggc cgtatgttgg ggcgcgaaac ggactccgtt ggaggaaagt cttgcttcga
     1621 tgattaacgg cggcggcaaa gacaagcccg atgagcatat cgatgccgtt aaaactcttt
     1681 ataaaagcct atacaacggt caataccaaa aacctgacgg caaagaaaaa ttctaccttt
     1741 taggtttatc gcccaattcc gcgcgcattg tcgtccgctt ttggcatgaa accaccgttg
     1801 ccgccttatc agaaagtatt gcggcgtggt atgacgattt gcaaatggtg cgcggcgaaa
     1861 actcgccata ccccgaatat atgccgctac cgcgcctgct gggtaatttg gtgttggacg
     1921 gcaaaatgga aaacctgcca tctgacctga ttgcccaaat aaccgatgcc gcgctcaaca
     1981 accgtgtttt acccgtcagc ctgttgcagg ctgctttgcg gcgcaacaag gcggaacaga
     2041 aaattaccta tggcagagca agtctgctta aagcctatat caatcgcgca atccgtgcgg
     2101 gtcgtctgaa aaacatgaag gagctaacta tgggcctaga tagaaaccgt caagacatcg
     2161 gctatgtgct ggggcggctg tttgccgtgc tggaaaaaat acaagccgag gccaatcccg
     2221 gcttgaacgc caccattgcc gaccgctatt tcggttcggc aagcagcaca ccgattgccg
     2281 tattcggcac actgatgcgc ttgttgccgc accatttgaa caaactggaa tttgaaggac
     2341 gtgccgtaca actgcaatgg gaaatccgcc agattttgga acattgtcag agatttccta
     2401 accatttgaa tttggaacag caaggcctat ttgccatcgg ttactaccac gaaacccaat
     2461 tcctgtttac caaagacgca ttgaaaaacc tgttcaacga agcgaaaacc gcataaggag
     2521 caaatgctat gactattgaa aaacgctacg actttgtctt tttatttgat gtgcaagacg
     2581 gcaatcccaa cggcgatcct gacgcaggta acctgccgcg tatcgacccg caaaccggcg
     2641 aaggtttggt aactgatgtt tgcctgaaac gcaaagtccg caactttatc caaatgactc
     2701 aaaatgacga acatcacgac atctttatcc gcgaaaaagg cattttgaac aacctgattg
     2761 acgaagccca cgagcaggaa aacgtaaaag gcaaagaaaa aggcgagaaa accgaagctg
     2821 cccgccaata catgtgcagc cgttattacg acatccgcac atttggcgca gtgatgacta
     2881 ccggcaaaaa tgcaggacaa gtacgcggtc ccgtgcaact gactttttct cgctctattg
     2941 atcccatcat gaccttggaa cacagcatta cccgcatggc ggttaccaac gaaaaagatg
     3001 ccagtgaaac cggcgacaac cgtacaatgg gtcgcaaatt caccgtcccc tacggtctat
     3061 accgctgcca tggcttcatt tctacccatt ttgccaaaca aacaggcttt tccgaaaacg
     3121 atttagagct gttttggcag gcacttgtca atatgtttga ccacgaccat tccgccgcac
     3181 gcggacaaat gaacgcacgc gggctctatg tgtttgaaca cagcaataat ctaggtgatg
     3241 cgcctgctga tagtctgttc aaacgcattc aggtagtcaa aaaggacggt gtagaagtag
     3301 taaggagttt tgacgattat cttgtcagcg tagacgataa gaatcttgaa gaaaccaagc
     3361 tgttgcgtaa attaggcgga ggctctgtgg gccccaagaa gaagcggaag gtgctcgagc
     3421 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3481 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3541 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3601 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3661 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3721 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3781 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3841 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3901 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3961 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4021 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     4141 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     4201 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     4261 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     4321 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     4381 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     4441 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4501 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4561 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4621 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     4681 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4741 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4801 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4861 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4921 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4981 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5041 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5101 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5161 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     5221 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5281 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5341 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5401 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5461 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5521 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5581 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5641 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5701 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5761 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5821 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5881 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5941 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     6001 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6061 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6121 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     6181 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     6241 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     6301 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     6361 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     6421 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     6481 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     6541 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     6601 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     6661 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     6721 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     6781 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     6841 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     6901 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     6961 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     7021 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     7081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     7141 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     7201 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     7261 gcggcccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     7321 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     7381 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     7441 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     7501 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     7561 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     7621 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     7681 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     7741 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     7801 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     7861 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     7921 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     7981 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     8041 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     8101 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     8161 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     8221 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     8281 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     8341 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     8401 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     8461 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     8521 cggcgt
//