LOCUS       Exported                8927 bp ds-DNA     circular SYN 22-DEC-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8927)
  AUTHORS   Doudna Lab
  TITLE     Direct Submission
  JOURNAL   Exported Dec 22, 2021 from SnapGene 4.2.11
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8927
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        671..689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             778..800
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             820..849
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             859..1959
                     /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"
     CDS             2014..2034
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2041..4998
                     /codon_start=1
                     /label=DpbCasX
                     /translation="MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEK
                     RRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASK
                     KIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHE
                     KLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASG
                     PVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLP
                     PQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENE
                     VDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENP
                     KKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSK
                     AVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGF
                     SIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYG
                     GGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIY
                     NKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVDRGENIPAVIALTDPEGCPLPEF
                     KDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSAR
                     DLFYHAVTHDAVLVFENLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLS
                     KTLAQYTSKTCSNCGFTITTADYDGMLVRLKKTSDGWATTLNNKELKAEGQITYYNRYK
                     RQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGH
                     EVHADEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA"
     primer_bind     3903..3922
                     /label=DpbCasX_Seq_Fwd1
     misc_feature    4054..4056
                     /label=Active site
     misc_feature    4345..4347
                     /label=Active site
     primer_bind     4448..4467
                     /label=DpbCasX_Seq_Fwd2
     primer_bind     complement(4486..4505)
                     /label=DpbCasX_Seq_Rev2
     primer_bind     4602..4634
                     /label=DpbCasX_NLS_Fwd_InFusion
     misc_feature    4843..4845
                     /label=Active site
     primer_bind     complement(5085..5104)
                     /label=DpbCasX_Seq_Rev1
     terminator      5138..5185
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(5551..5579)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        5692..5796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5797..6657
                     /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"
     rep_origin      6828..7416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    7602..7744
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(7846..8037)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag
       61 caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa
      121 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
      181 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
      241 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
      301 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
      361 gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
      421 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
      481 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
      541 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
      601 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
      661 cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc
      721 tccggagagc tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt
      781 gtttaacttt aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatcac
      841 catcaccatg gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc
      901 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
      961 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
     1021 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
     1081 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
     1141 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
     1201 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
     1261 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
     1321 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
     1381 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
     1441 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
     1501 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
     1561 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
     1621 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
     1681 gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     1741 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     1801 gatccacgta ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc
     1861 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     1921 cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac
     1981 aacaataaca ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca
     2041 atggagaagc gtattaacaa gattcgtaag aagttatcgg ctgataatgc cacaaaacct
     2101 gtaagccgct caggacctat gaaaacgctt ttagttcgtg ttatgacaga tgatctgaaa
     2161 aaacgtcttg agaagcgtcg taaaaagccg gaggtcatgc ctcaagtcat ttcgaataat
     2221 gccgccaata atttacgtat gcttcttgat gactatacaa agatgaaaga agcaatcctg
     2281 caagtctact ggcaagagtt caaagatgat catgtaggtt tgatgtgtaa gtttgcacag
     2341 cccgctagta aaaagatcga ccaaaacaag ttaaagccag agatggacga gaaagggaat
     2401 cttaccactg ccgggttcgc ttgctctcaa tgcggccaac cgctgttcgt atataagctg
     2461 gagcaagttt cagaaaaggg taaggcatac acaaattatt tcggacgttg caacgtcgct
     2521 gagcacgaga agcttattct tttagcccag ctgaaaccag agaaggactc tgatgaagcg
     2581 gttacctact ccttgggaaa gttcggacag cgcgctcttg acttttattc catccacgtg
     2641 acaaaggaaa gtacacaccc cgtcaaacca ctggcccaaa ttgcagggaa ccgctacgcg
     2701 agcgggccag tgggaaaagc attgtcagac gcttgtatgg gcacgattgc ttcctttctt
     2761 agtaaatacc aggatattat cattgaacat caaaaggtag ttaaggggaa ccagaagcgt
     2821 ctggaaagct tacgtgaact tgcagggaaa gagaaccttg agtacccatc ggtcacattg
     2881 cctccgcagc cgcacacaaa ggagggtgtc gatgcttata acgaggtgat tgcacgcgta
     2941 cgcatgtggg taaatttgaa tctgtggcag aagttgaaac tgtcgcgtga tgacgccaaa
     3001 cctcttttac gcctgaaggg cttcccctca tttccagtcg tagagcgccg tgaaaatgaa
     3061 gtagactggt ggaatactat caacgaagta aaaaagctga ttgacgcgaa gcgcgacatg
     3121 ggacgcgtgt tttggagcgg cgttactgct gaaaagcgca acaccatcct tgaaggatac
     3181 aactatttac ccaacgagaa cgatcataaa aagcgcgagg gcagtttaga gaatccgaag
     3241 aagccggcca aacgtcagtt tggcgacctt ttattatacc tggaaaagaa atatgcgggt
     3301 gactggggca aagtgttcga tgaagcctgg gagcgcattg acaagaaaat cgctggtctg
     3361 acgtcgcaca ttgagcgcga ggaagctcgt aacgcagaag acgcccaatc caaagcggtc
     3421 ctgacagact ggctgcgcgc taaggcgtcc tttgttcttg agcgtttaaa agagatggac
     3481 gaaaaggaat tctatgcctg tgagattcaa ttacaaaagt ggtatggtga cttacgcggg
     3541 aacccctttg ctgtagaggc tgagaatcgt gtagtggaca tttcaggttt cagcatcgga
     3601 tcggatggac attctatcca atatcgtaac cttcttgcat ggaaatattt ggagaatggt
     3661 aagcgcgagt tctatctgct tatgaactac gggaaaaaag gacgtattcg cttcaccgat
     3721 ggtacagaca ttaaaaaaag tggcaaatgg caagggttgt tatatggggg tggcaaagca
     3781 aaggttatcg atttaacgtt tgatcccgat gatgagcagc tgattatctt gcctttagct
     3841 ttcggaaccc gccagggccg tgaatttatc tggaacgact tgctttctct tgagacagga
     3901 cttatcaaac ttgcgaatgg acgcgtgatc gaaaagacta tttacaataa aaagatcggt
     3961 cgcgatgagc cggccctgtt tgtcgcctta acattcgagc gtcgcgaagt tgttgatccg
     4021 tcaaatatca agccggtaaa cttaatcggg gtcgatcgtg gtgaaaatat cccggcagta
     4081 atcgctctta cagacccaga aggctgcccc ttacccgagt tcaaggactc atccggcggt
     4141 ccaacagaca tccttcgcat tggcgaagga tataaagaga aacagcgtgc aatccaggca
     4201 gcaaaagaag tggaacaacg tcgcgccgga ggctacagcc gcaaatttgc gtcaaaatcg
     4261 cgtaacttgg cagatgatat ggtccgcaac agcgcccgtg atttgtttta ccacgcggtg
     4321 acacatgacg ctgttttagt ttttgagaac ttgtcacgtg ggttcggacg tcagggaaaa
     4381 cgcaccttca tgactgagcg ccaatacaca aaaatggaag actggttgac ggctaaactg
     4441 gcttacgagg ggcttactag taaaacgtat ttgtcgaaaa ctcttgctca gtacacctct
     4501 aaaacttgtt ctaactgcgg gttcacgatt accacagcgg actacgatgg catgctggta
     4561 cgccttaaaa agacgagcga cgggtgggca acgacattga acaataaaga acttaaggcc
     4621 gagggacaaa tcacctacta taatcgttat aagcgccaga cggtagaaaa agagttaagc
     4681 gccgaacttg accgcctgag cgaggagagt gggaacaacg acatttcaaa atggactaag
     4741 gggcgccgtg acgaggcttt gtttttgctg aagaaacgtt tctcccatcg tcccgtacaa
     4801 gagcagttcg tttgcttaga ttgtggccat gaagttcatg ctgatgaaca ggctgcttta
     4861 aatatcgcgc gttcctggtt gtttcttaat agtaactcaa ctgagttcaa atcatataag
     4921 tcagggaaac agccgtttgt gggagcatgg caggcttttt ataaacgccg cctgaaggag
     4981 gtctggaagc cgaatgctta acattggaag tggataacgg atccgcgatc gcggcgcgcc
     5041 acctggtggc cggccggtac cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa
     5101 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     5161 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atccacagga
     5221 cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga
     5281 ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca
     5341 tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga
     5401 tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg
     5461 tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc
     5521 gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa
     5581 catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5641 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5701 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5761 tgataaatgc ttcaataaca ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5821 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5881 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5941 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6001 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     6061 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6121 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6181 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6241 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6301 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     6361 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6421 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6481 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6541 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6601 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6661 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6721 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6781 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6841 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6901 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6961 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7021 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7081 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7141 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7201 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7261 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7321 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7381 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7441 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7501 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7561 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     7621 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     7681 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     7741 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7801 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7861 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     7921 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     7981 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     8041 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     8101 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     8161 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     8221 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     8281 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     8341 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     8401 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     8461 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     8521 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     8581 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     8641 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     8701 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     8761 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccagtg
     8821 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     8881 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgcc
//
