LOCUS       sequence-238616-        7158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas2 cloned in pET16b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7158)
  AUTHORS   Nunez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA
  TITLE     Cas1-Cas2 complex formation mediates spacer acquisition during 
            CRISPR-Cas adaptive immunity.
  JOURNAL   Nat Struct Mol Biol. 2014 Jun;21(6):528-34. doi: 10.1038/nsmb.2820. 
            Epub 2014 May 4.
  PUBMED    24793649
REFERENCE   2  (bases 1 to 7158)
  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/nsmb.2820";
            journalName: "Nat Struct Mol Biol"; date: "2014-06"; volume: "21"; 
            issue: "6"; pages: "528-34"
COMMENT     Sequence Label: pET16b-Cas2
FEATURES             Location/Qualifiers
     source          1..7158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        86..104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    105..129
                     /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             145..167
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             469..489
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             502..1602
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1576..1599
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1675..1692
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1745..1763)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1759..1806
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1981..1986,1987..2003,2004..2009))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2036..2054)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2122..2226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2227..2295,2296..3087)
                     /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(2445..2464)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3258..3846
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3747..3766
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4000..4017
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4032..4171
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4097..4116)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4257..4279
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4273..4464)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6819..6838
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6858..6935)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(7141..7158,1..2)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
       61 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
      121 aacaattccc ctctagagag ctcttttgtt taactttaag aaggagatat acatatgatg
      181 agtatgttgg tcgtggtcac tgaaaatgta cctccgcgct tacgaggcag attagccatc
      241 tggttgttgg aggtacgtgc aggggtatat gtaggtgatg tatccgcaaa aattcgtgaa
      301 atgatctggg aacaaatagc tggactggcg gaagaaggca atgtagtgat ggcatgggca
      361 acgaatacgg aaacgggatt tgagttccag acatttgggt taaacaggcg taccccggta
      421 gatttggatg gtttaaggtt ggtgtctttt ttacctgttg gttcttctga aaacctgtac
      481 ttccaatccg gatccggcgg catgaaaatc gaagaaggta aactggtaat ctggattaac
      541 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga
      601 attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
      661 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa
      721 tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt
      781 acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg
      841 ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc
      901 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa
      961 gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
     1021 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg
     1081 accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc
     1141 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg
     1201 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt
     1261 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac
     1321 aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
     1381 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg
     1441 aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac
     1501 atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc
     1561 ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac
     1621 aacaacaata acaataacaa caacctcggg atcgagggtt cttctaaatc ttctcaccat
     1681 caccatcacc attgagcggc cgctgctaac aaagcccgaa aggaagctga gttggctgct
     1741 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1801 tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca
     1861 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagtgcat
     1921 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
     1981 attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc
     2041 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2101 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2161 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2221 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2281 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2341 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2401 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2461 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2521 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2581 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2641 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2701 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2761 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2821 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2881 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2941 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3001 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3061 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3121 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3181 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3241 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3301 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3361 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3421 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3481 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3541 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3601 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3661 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3721 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3781 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3841 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3901 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3961 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4021 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4081 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4141 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4201 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4261 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4321 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4381 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4441 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4501 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4561 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4621 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4681 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4741 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4801 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4861 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4921 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     4981 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     5041 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     5101 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     5161 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     5221 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     5281 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     5341 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     5401 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     5461 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     5521 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     5581 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     5641 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     5701 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5761 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa actgtcgtgc cagctgcatt
     5821 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5881 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5941 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6001 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     6061 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6121 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6181 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6241 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6301 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6361 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6421 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6481 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6541 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6601 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6661 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6721 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6781 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6841 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6901 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6961 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     7021 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7081 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7141 ggtgatgtcg gcgatata
//
