LOCUS       sequence-215734-        6009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express enterococcus faecalis Cas2 protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  AUTHORS   Xiao Y, Ng S, Nam KH, Ke A
  TITLE     How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated
            spacer integration.
  JOURNAL   Nature. 2017 Oct 5;550(7674):137-141. doi: 10.1038/nature24020. Epub
            2017 Sep 4.
  PUBMED    28869593
REFERENCE   2  (bases 1 to 6009)
  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: 
            "10.1038/nature24020"; journalName: "Nature"; date: "2017-10-5- 5"; 
            volume: "550"; issue: "7674"; pages: "137-141"
COMMENT     Sequence Label: His6-Twin-Strep-SUMO-pET28a-EfaCas2
FEATURES             Location/Qualifiers
     source          1..6009
                     /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(497..790)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(join(791..814,815..850,851..874))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(887..904)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(914..931)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             950..972
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    987..1011
                     /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(1011..1030)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1012..1030)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1114..1133)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1339..1416
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1417..2499
                     /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(1436..1455)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3308..3499
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3493..3515)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3601..3740
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3656..3675
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3755..3772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3926..4514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4006..4025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4625..4644)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4636..5451
                     /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             4636..5451
                     /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(4709..4728)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5543..5998)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5680..5701)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5892..5911
                     /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 gagttattct tctcctaaaa
      181 agacaatccg ttcatctgaa ttagcaactc gattgtcttg ttctccatgc aaataaatca
      241 ttcgagaaaa ttgtttttct gttatattta atagtgtaat tagtcctctt tgcggattat
      301 tctgctttaa gcgagccaac attgctttat tagcagtatc attcaacaaa attttgctgt
      361 aaaccgaaaa ttgatgcata ataaatcctt cattaatcaa aaatttccgg aacttccgat
      421 acgctttccg gtcactggct gtgtccgtcg gcatatcaaa cattaataat agacgcatat
      481 atcgataact catggatcca ccaatctgtt ctctgtgagc ctcaataata tcgttatcct
      541 ccatgtccaa atcttcaggg gtctgatcag cttggattct aataccgtcg tacaagaatc
      601 ttaaggagtc catttcctta ccctgtcttt tagcgaacgc ttccatcagc cttcttaaag
      661 gagtggtctt tttgatcttg aagaagatct ctgaagatcc atcggacacc tttaaattga
      721 tgtgagtctc aggcttgact tctggcttga cctctggctt agcttcttga ttgacttctg
      781 agtccgacat tttctcaaac tgagggtgtg accatgcact accacctgaa ccaccaccgc
      841 taccaccacc tttttcaaac tgcggatggc tccagctagc catatggctg ccgcgcggca
      901 ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat ctccttctta
      961 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     1021 agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg
     1081 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     1141 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
     1201 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
     1261 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
     1321 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     1381 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     1441 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     1501 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     1561 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     1621 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     1681 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     1741 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     1801 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     1861 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     1921 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     1981 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     2041 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     2101 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     2161 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     2221 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     2281 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     2341 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     2401 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2461 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     2521 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     2581 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     2641 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     2701 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     2761 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     2821 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     2881 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     2941 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     3001 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     3061 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     3121 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     3181 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaacca gtatcgtgag
     3241 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     3301 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     3361 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     3421 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     3481 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3541 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3601 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     3661 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     3721 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3781 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3841 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3901 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3961 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4021 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4081 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4141 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4201 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4261 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4321 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagraca gtatttggta
     4381 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4441 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4501 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4561 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     4621 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     4681 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     4741 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     4801 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     4861 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     4921 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     4981 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     5041 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     5101 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     5161 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     5221 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5281 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5341 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5401 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     5461 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5521 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     5581 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5641 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5701 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5761 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5821 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     5881 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     5941 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6001 cattcgcca
//
