LOCUS       sequence-215730-        6471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express enterococcus faecalis Cas1 protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  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 6471)
  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-SUMO-pET28a-EfaCas1
FEATURES             Location/Qualifiers
     source          1..6471
                     /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(1043..1336)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(1349..1366)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1376..1393)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1412..1434
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1449..1473
                     /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(1473..1492)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1474..1492)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1576..1595)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1801..1878
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1879..2961
                     /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(1898..1917)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3770..3961
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3955..3977)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4063..4202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4118..4137
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4217..4234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4388..4976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4468..4487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5087..5106)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5098..5913
                     /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             5098..5913
                     /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(5171..5190)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6005..6460)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6142..6163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6354..6373
                     /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 gagtgcggcc gctcatatcc
      181 caaactctgg aactccttcc tcctcttgat tcagaacttt tactacatgt tttgtataat
      241 cggtagcaat attggttaaa aacatttgtt ttttcttgta catatacgta ttcataaaca
      301 aagcaaataa cttacgtttc attatcggaa atgcttcttt tctattctca taaatgattt
      361 gatctaccaa tggacgaaaa ggctccataa gatcacttgc taagttaaag tcattaaatt
      421 ggttggcatg ttttagtcct agttgtgtaa aacaacctgt ctggactaac tctcgagcaa
      481 aaatacttaa taacaacgta taaccatagt ttaaaccagc attaatgtca ttttcttgtt
      541 cacgtgtaaa atcattgcca aagagagtat taaaataact tcgagctgca tgaccttctc
      601 gattggctgg atcaaacaga cgtaattcag cgcgcatagc taacaaagca gcagcctttt
      661 ggtcatagtc tcgttgcgct aagtgtaaac tctgattggt gattttttgt gcaatgatcg
      721 ctgtccaaac ttgacctttt cgttcttccg tccaagccaa ctgacgtgtc aattgcaagc
      781 tactatcatg ccgtccataa aagggcaaaa ttttaccaat tggcaaacgc ttatcatcgc
      841 aaaataacac taatattttt tcatcaatta atcgttttaa tagcatcgtg gttaacgaaa
      901 tgtccgttgt ttccaacaac aaaacatcaa tctcagacaa atgaattaat tcttggtgat
      961 caatggcttt aaaaacaaga tgattgtttt tataggataa ttttgaatgc ttgttaacca
     1021 ctaccgttcg ccagcccatg gatccaccaa tctgttctct gtgagcctca ataatatcgt
     1081 tatcctccat gtccaaatct tcaggggtct gatcagcttg gattctaata ccgtcgtaca
     1141 agaatcttaa ggagtccatt tccttaccct gtcttttagc gaacgcttcc atcagccttc
     1201 ttaaaggagt ggtctttttg atcttgaaga agatctctga agatccatcg gacaccttta
     1261 aattgatgtg agtctcaggc ttgacttctg gcttgacctc tggcttagct tcttgattga
     1321 cttctgagtc cgacatgcta gccatatggc tgccgcgcgg caccaggccg ctgctgtgat
     1381 gatgatgatg atggctgctg cccatggtat atctccttct taaagttaaa caaaattatt
     1441 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     1501 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1561 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1621 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1681 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1741 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1801 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1861 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1921 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1981 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     2041 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     2101 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     2161 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     2221 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     2281 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     2341 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     2401 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     2461 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     2521 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     2581 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     2641 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     2701 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2761 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2821 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2881 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2941 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     3001 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     3061 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     3121 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     3181 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     3241 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     3301 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     3361 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     3421 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     3481 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     3541 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     3601 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3661 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     3721 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     3781 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     3841 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     3901 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3961 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4021 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     4081 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     4141 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     4201 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4261 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4321 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4381 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4441 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4501 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4561 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4621 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4681 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4741 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4801 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4861 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4921 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4981 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5041 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     5101 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     5161 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     5221 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     5281 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     5341 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     5401 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     5461 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     5521 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     5581 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     5641 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     5701 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     5761 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     5821 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     5881 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     5941 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6001 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     6061 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     6121 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     6181 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     6241 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     6301 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     6361 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     6421 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
