LOCUS       sequence-394539-        7781 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For protein purification of Csx30.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7781)
  AUTHORS   Strecker J, Demircioglu FE, Li D, Faure G, Wilkinson ME, Gootenberg 
            JS, Abudayyeh OO, Nishimasu H, Macrae RK, Zhang F
  TITLE     RNA-activated protein cleavage with a CRISPR-associated 
            endopeptidase.
  JOURNAL   Science. 2022 Nov 25;378(6622):874-881. doi: 
            10.1126/science.add7450. Epub 2022 Nov 3.
  PUBMED    36423276
REFERENCE   2  (bases 1 to 7781)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.add7450"; journalName: "Science"; date: 
            "2022-11-25- 25"; volume: "378"; issue: "6622"; pages: "874-881"
COMMENT     Sequence Label: SUMO_TwinStrep_Csx30
FEATURES             Location/Qualifiers
     source          1..7781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    403..542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(468..487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     628..650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(644..835)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2147..3229)
                     /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     3191..3210
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3230..3307)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3513..3532
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3616..3635
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3616..3634
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3635..3659
                     /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             3674..3696
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3715..3732
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3742..3759
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(3772..3795,3796..3831,3832..3855)
                     /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             3856..4149
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(5897..5915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5911..5958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6133..6138,6139..6155,6156..6161))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6188..6206)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6274..6378
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6379..6447,6448..7239)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6597..6616)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7410..7781,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
      421 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
      481 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
      541 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
      601 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
      661 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
      721 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
      781 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
      841 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
      901 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
      961 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     1021 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     1081 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     1141 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     1201 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     1261 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc
     1321 ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag
     1381 ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg
     1441 tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc
     1501 accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa
     1561 cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat
     1621 cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc
     1681 tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag
     1741 aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag
     1801 cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1861 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1921 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1981 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     2041 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     2101 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     2161 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2221 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     2281 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     2341 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     2401 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     2461 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     2521 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     2581 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     2641 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     2701 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     2761 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     2821 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     2881 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2941 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     3001 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3061 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     3121 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     3181 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     3241 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     3301 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     3361 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     3421 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     3481 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     3541 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     3601 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     3661 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat
     3721 catcatcatc acagcagcgg cctggtgccg cgcggcagcc atatggctag ctggagccat
     3781 ccgcagtttg aaaaaggtgg tggtagcggt ggtggttcag gtggtagtgc atggtcacac
     3841 cctcagtttg agaaaatgtc ggactcagaa gtcaatcaag aagctaagcc agaggtcaag
     3901 ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatggatc ttcagagatc
     3961 ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt cgctaaaaga
     4021 cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat ccaagctgat
     4081 cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca cagagaacag
     4141 attggtggat ccaataccac aacatataac actacgactg atgctttgct ggagtggggt
     4201 aaggtgtact tccagaaaga agatttctcc gagtttctgg acaatttgga ggcgtacatc
     4261 agcgatgcgg gcgacagcct caaggacgag cttgagtctg gtgttgaaaa gctggttctg
     4321 ggtattaaaa gcgcggaggc ggttatcttc ggcgaagcgg tgatcggcac caccccggaa
     4381 aacgaagcat ggtatgacgc cgaagagtcg ttcctgaccc tggactgcgc agtgtggctg
     4441 tcacaggcat tggaccgtgt cgtgcgtcgt caggacgcga gcttggcgga ttcgctgatc
     4501 gcgcggctgg atgaggccat taaccgcgtg gctgaaaaac tgtacgcgga caacctgtcc
     4561 ccgctgcgct tttccagctt gaacgagatc cgtcgtagcg cgctggaagc taccgatgag
     4621 aaataccatt atctgttccc gtggcatggt gcagcgtgcg atgttgatga gaacattctt
     4681 ctgatcttaa ccgaagagta ccacctgata ggcgcggata aagcgggtgc aaatctgagc
     4741 gaagaactgc gtggtgacct gccgtttatc tttgcagaac tggagcgtga cgaggtgctg
     4801 cgcgcatatg ttgaaaagga gaacgcgctg tctctggctt tggagaatac catgcgtgaa
     4861 cattgggcat ttggcctgct agaggcggct agagatgagg gctataatca cccgtatccg
     4921 gctgatgtgg gtatgcgtat tcatcaggtt gcccgcgcgg ttttttcgca aacgaacctg
     4981 tccccggcgg aacgcctggc tgtagccatc gctggagcgt gcttcacccc ggaaatcagc
     5041 gaggaccgtc gtttggagat cctcttggac tgtgaagaac gtgtctgtga aattgaagca
     5101 ccgactggtg acgacacgag cgtacgtgtt atcaaggatc tgaaggccct ggctgatcat
     5161 cgtgtccgcc acgaaatccc agccgagtcc ctcgtgagcc tgtggtttga acagattgaa
     5221 gccgcaggca cggacttcga taccaaaacc cctatggatg agcttgtgct gcgtatgctg
     5281 tccgataacg tgatcaccct gtcggtggat cgtaaagccg cgagccaaac cgagacagac
     5341 gacgttaagc cgcagaaagg taaaatcatc ccgttcccgg ttccggatat cgcgaacgat
     5401 gaggtggagt accagaaagc tgtgggtatg aaaaaggaca aaaaggccgc gaacgactcc
     5461 aaagtgaagt ttccgggtct gttggagatc caaggttgcc gtgatggcga taaggcgatt
     5521 ctattggagg acactgacga cgcagccgcg aaccaccgca aactatttag tattctgaag
     5581 gcgggtaagc tgaacagcgc gttcttcatc caatctgacg acggcgagtg ggttgaatct
     5641 gagagtaagc cgaccatgga agacaatcgt attattttgc acgatagcca ccacagcagc
     5701 ttcgtttgga ttctcgacac gggtagcatg cagctgcgcc aaagcgttaa gtgcgttaag
     5761 gatgcgctga ataaaaagac gggctctgcg aaaaaattga agccaaaaac catgatcgtg
     5821 tgggtcacca ttccgcaaga aggctaatga gcggccgcac tcgaggcccg aaaggaagct
     5881 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     5941 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc aagaggcccg
     6001 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
     6061 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
     6121 ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga
     6181 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6241 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6301 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6361 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6421 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6481 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6541 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6601 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     6661 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6721 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6781 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6841 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6901 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     6961 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7021 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7081 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7141 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7201 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7261 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7321 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     7561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7621 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac t
//
