LOCUS       Exported                8613 bp ds-DNA     circular SYN 06-OCT-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8613)
  AUTHORS   Yiping Qi Lab
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Oct 6, 2021 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8613
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        1..138
                     /label=Spn promoter
     CDS             139..1149
                     /label=SpnR
     rep_origin      1216..1889
                     /direction=RIGHT
                     /label=pUC origin
     terminator      2319..2362
                     /label=rrnB T1 terminatior
     primer_bind     2429..2444
                     /label=M13 (-20) forward primer
     misc_recomb     2461..2543
                     /label=attL1
     protein_bind    2544..2560
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2567..2587
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2594..3181
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     misc_feature    3194..3478
                     /label=95aa linker
     CDS             3491..3511
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3518..4105
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     gene            4184..4234
                     /label=N-terminal NLS
                     /note="N-terminal NLS"
     CDS             4235..7984
                     /codon_start=1
                     /label=dMb2Cas12a
                     /translation="LFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY
                     QKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDGLQKQLKDLQAVLRK
                     ESVKPIGSGGKYKTGYDRLFGAKLFKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFS
                     TYFTGFHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINE
                     LTASGLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKSERIAKLRPL
                     HKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYTDVFAKVQSLFDGFDDHQKDGIYV
                     EHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFI
                     KGVHSLASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFL
                     ERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFY
                     GEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVI
                     LQKDGCYYLALLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAKSNLDYY
                     NPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYRDL
                     SDFYREVEPQGYQVKFVDINADYIDELVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFK
                     ALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDI
                     IKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIARGERHLLY
                     LTVINSKGEILEQRSLNDITTASANGTQVTTPYHKILDKREIERLNARVGWGEIETIKE
                     LKSGYLSHVVHQINQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHL
                     VLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKP
                     RYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTDKAKNSRQKWAICSHGDKRYVY
                     DKTANQNKGAAKGINVNDELKSLFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTL
                     LALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNEL
                     KNSDDLNKVKLAIDNQTWLNFAQNR"
     misc_feature    6797..6816
                     /label=Mb2Cas12aD864A-R
     misc_feature    6817..6835
                     /label=Mb2Cas12aD864A-F
     misc_feature    6821..6823
                     /label=D864A
     misc_feature    7985..8032
                     /label=Nucleoplasmin NLS
                     /note="Nucleoplasmin NLS"
     misc_feature    8033..8035
                     /label=stop
                     /note="stop"
     misc_feature    8061..8338
                     /label=NOS term
     misc_recomb     8360..8483
                     /label=attR5
     primer_bind     8524..8540
                     /label=M13 reverse primer
     promoter        8612..8613
                     /label=Spn promoter
ORIGIN
        1 tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
       61 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
      121 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      181 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
      241 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
      301 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg
      361 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
      421 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      481 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      541 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      601 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      661 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      721 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      781 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      841 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      901 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      961 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     1021 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     1081 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     1141 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt agaaacgcaa
     2161 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     2221 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     2281 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     2341 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgctt
     2401 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2461 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
     2521 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgatgccca aaaaaaaaag
     2581 aaaggtctcg tcacagctcg tcaagagcga actggaagag aagaaatcgg aattgcgcca
     2641 caaactcaaa tacgtccctc atgagtacat cgaactcata gaaatcgcca gaaattctac
     2701 ccaagaccgg atacttgaaa tgaaggtcat ggaatttttt atgaaggtgt atgggtatag
     2761 aggaaagcac ctcggtggca gtaggaagcc cgatggcgcg atctacaccg tcggtagtcc
     2821 gattgactat ggagtgatag tcgacacaaa agcgtatagt ggagggtata acctgcccat
     2881 tggacaagca gatgaaatgc agcggtacgt ggaagaaaac caaacacgca ataaacatat
     2941 aaatcctaac gaatggtgga aggtttatcc ctcctccgtt acagaattca agtttttgtt
     3001 cgtctcgggc cacttcaagg gcaactacaa ggcccagctt actcggttga atcacatcac
     3061 aaattgcaat ggggcagtct tgagtgtgga ggaacttttg atcgggggtg aaatgatcaa
     3121 ggccggtacg ctgacgttgg aggaggttcg gcggaagttt aataatggcg aaataaactt
     3181 taccggtgct agcgggtctg ggtcaggttc cggctcgatt actcggacaa caaatccacg
     3241 gaacgtcgtt cctaaaatct atatgtcagc agggtcgata ccactgacga cgcatataac
     3301 caatagcatt cagcctaccc tgtggacgat tgggtccatc aacggtgtcg cccccttggc
     3361 gaagtctata aaacttggga tacccgtcac tggatcagca tacaccgacc agacgactgc
     3421 tatggttagg aagaaagtgt ctgtttttat gggaagtggt tcaggatccg gctcaagtct
     3481 gcagactagt ccaaaaaaaa agcggaaggt tagttcacaa ctggtgaagt ctgaacttga
     3541 ggagaagaag agcgagttga ggcacaagtt gaagtatgtc ccccatgaat acatcgagtt
     3601 gattgagatt gctcgcaaca gcacccagga ccggattctc gaaatgaagg tcatggaatt
     3661 ttttatgaaa gtgtacgggt accgcggaaa gcatctcggg ggaagtcgca aacctgacgg
     3721 ggctatctat acggtgggtt ctcctataga ctacggggtc attgtggata cgaaggccta
     3781 tagtggcgga tacaatctgc caattggaca agctgatgaa atgcagaggt acgttgaaga
     3841 gaatcagacc cgcaacaaac atataaaccc aaatgaatgg tggaaggtgt accccagcag
     3901 tgtcaccgaa ttcaaatttc tgtttgtgag tgggcatttc aaaggaaact ataaagcaca
     3961 acttacgaga ctgaaccata ttaccaattg taacggcgct gtgttgagtg tcgaggagct
     4021 gctcataggg ggagaaatga tcaaagcagg cacattgact ctcgaggagg tgcggcggaa
     4081 gtttaacaat ggggaaatca acttcggtgg tggtggttct ggaggtggag gctctggagg
     4141 aggaggcagc ggtggaggcg ggtctggagg tggtggttct accatggctc ctaagaagaa
     4201 gcggaaggtt ggtattcacg gggtgcctgc ggctctgttt caagatttta cacatctgta
     4261 cccgctgagt aaaacagtgc ggttcgagct gaaacccata ggaaggaccc tcgagcacat
     4321 ccacgcgaag aattttctga gccaggatga aactatggct gatatgtatc aaaaagttaa
     4381 ggtcattttg gacgactatc atcgcgattt tattgccgac atgatgggag aggtgaaact
     4441 cacgaagctt gctgaatttt acgacgtcta tctgaagttc aggaaaaatc ctaaggacga
     4501 tgggctgcaa aaacagctta aagaccttca agctgtcctt cggaaggaat cggtgaagcc
     4561 tatagggtca ggtgggaagt acaaaacagg ctacgataga ctctttgggg caaaactctt
     4621 caaagatgga aaagagttgg gtgacctcgc aaaattcgtt atagcccaag aaggtgagtc
     4681 ttctccgaag ctggctcatc ttgctcattt tgagaagttc agcacgtatt ttactggatt
     4741 tcacgataat cggaagaata tgtactcgga tgaagacaag catactgcaa tagcgtacag
     4801 gctcatccat gagaatttgc cgagattcat cgacaatctg caaatcttga caacaatcaa
     4861 acaaaagcat agcgccctct atgatcagat aatcaacgag ctcacggcct ccgggctcga
     4921 cgtctccttg gcttctcatc ttgacgggta tcacaagctc cttacacaag aggggatcac
     4981 ggcatacaac aggatcatag gagaggtgaa tggatataca aataagcata accagatatg
     5041 ccacaagagc gagcgcatag cgaaacttag acccttgcac aagcaaatcc tttctgacgg
     5101 aatgggagtg tcattccttc cgtctaagtt cgcggatgat agtgagatgt gccaagcggt
     5161 caacgaattt tatcgccatt atactgacgt gttcgcaaag gtgcaaagtc tctttgacgg
     5221 atttgatgat caccagaaag acgggatcta tgttgaacac aaaaacctta atgaactgag
     5281 caaacaggcg ttcggcgact ttgctttgct ggggagggtc cttgatggat actacgtgga
     5341 cgttgtcaat ccggagttca atgagcggtt cgcaaaggcc aagactgaca atgcgaaagc
     5401 caagcttaca aaagaaaagg acaaattcat taaaggagtc cactcactgg cttccctcga
     5461 acaagcaata gaacaccata cagctagaca cgacgatgag agtgttcaag ccggaaaact
     5521 tggccagtac ttcaaacacg gtttggcggg ggttgacaac ccgattcaga aaattcacaa
     5581 taaccattcg acgattaaag ggtttctgga aagggaaagg cctgctgggg aacgggcgct
     5641 cccgaagatc aagtcaggaa aaaacccaga aatgacacag ctcaggcagc tgaaggaact
     5701 tttggacaac gcattgaatg tggcgcactt cgctaagctg ctgacaacta aaacaacctt
     5761 ggacaaccag gatggaaatt tttacgggga gtttggggtg ctttacgacg agctggctaa
     5821 aattccaact ctctacaata aggttagaga ttatctctct caaaagccct tttctaccga
     5881 aaagtataag ctcaacttcg gcaatccgac ccttctcaat gggtgggacc tgaacaaaga
     5941 gaaagataac tttggggtta tacttcagaa ggatggatgc tattacttgg cgcttcttga
     6001 taaggctcat aaaaaagttt tcgacaacgc ccctaacact ggtaagaacg tctaccaaaa
     6061 gatggtctac aaactgttgc ccggccccaa caaaatgctt cctaaagtgt ttttcgcaaa
     6121 atcgaatctc gactattata atccatctgc cgagctcctt gacaaatatg ctaaggggac
     6181 ccataaaaag ggtgataatt tcaacctgaa ggactgccac gcgcttatcg actttttcaa
     6241 agccgggata aataagcatc cggagtggca acattttggt tttaaatttt cgccaacgtc
     6301 gtcctatcgc gacctttccg atttctatag ggaagttgaa cctcaggggt accaggtcaa
     6361 atttgttgac attaatgcgg actacattga tgaattggtg gagcaaggga agctctacct
     6421 ctttcaaata tataacaaag atttctcgcc aaaagcgcat ggtaaaccga atcttcatac
     6481 cttgtacttt aaagcacttt tttcagaaga taacttggcg gacccgatct acaagctgaa
     6541 tggggaagct cagatcttct acaggaaagc ttcgttggac atgaacgaga ctaccataca
     6601 tcgcgcggga gaggtgcttg agaacaaaaa tcccgacaac ccgaaaaagc ggcaattcgt
     6661 ttacgacatc atcaaagaca aacggtacac gcaggacaaa tttatgctcc acgtccccat
     6721 taccatgaat tttggagtcc aaggcatgac cattaaggaa ttcaacaaaa aggtcaacca
     6781 aagtattcag caatacgatg aagtcaatgt cataggcata gctcggggag aaaggcatct
     6841 gttgtatctt accgtgatta actctaaggg tgaaatactg gagcaacggt cacttaacga
     6901 tataaccacg gcgtccgcga acggtacaca agtgaccact ccctaccaca aaatattgga
     6961 taaaagggag atagaacgct tgaatgcccg cgttggctgg ggtgagattg agaccatcaa
     7021 agagcttaaa tcgggatatt tgtctcacgt cgttcatcaa attaaccaac tcatgcttaa
     7081 gtacaatgca atcgttgtgc tcgaggacct gaactttggt ttcaaaagag ggaggttcaa
     7141 ggtggaaaaa caaatttacc agaactttga aaacgcgctt atcaagaaat tgaatcacct
     7201 tgttttgaaa gataaggcag atgacgaaat cgggtcgtat aaaaatgcac tccagttgac
     7261 aaataatttc acggatttga agtcgatcgg caagcaaaca gggttcctct tttatgtgcc
     7321 agcgtggaat acatcaaaaa ttgatccgga gacgggattt gtcgacttgc tgaagcctag
     7381 gtatgagaac attgcccaat ctcaggcctt tttcggcaaa ttcgataaaa tatgctacaa
     7441 cacagacaaa ggttattttg aatttcacat tgattacgcc aaatttacag ataaggcgaa
     7501 aaacagcaga cagaaatggg ctatctgttc tcatggggac aaacgctatg tctacgataa
     7561 gacggctaat caaaataaag gcgccgcaaa aggtattaat gtgaatgatg agctgaaaag
     7621 cttgtttgcc cgctaccata tcaatgataa acaaccaaac ttggtgatgg acatatgcca
     7681 gaacaatgac aaagaattcc acaagtcact catgtgcctg cttaaaaccc ttttggcgct
     7741 gcggtatagc aatgcatcta gcgatgaaga ctttattttg agtcccgtgg ccaacgacga
     7801 gggcgtgttt tttaattcag ccttggcgga cgatacgcag ccccagaatg cggacgcaaa
     7861 cggcgcgtac cacattgcac tgaagggact gtggcttctg aacgagctga aaaatagcga
     7921 cgacctgaat aaagtcaagt tggccattga caatcaaacc tggttgaatt tcgctcaaaa
     7981 tagaaagcgt cctgctgcca ccaaaaaggc cggacaggct aagaaaaaga agtgagacga
     8041 ctagtggcgg ccgccgacgt ccgatcgttc aaacatttgg caataaagtt tcttaagatt
     8101 gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca
     8161 tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt
     8221 cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa
     8281 attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttgatccccc ctcgacagct
     8341 tccggaaagg gcgaattcgc aactttgtat acaaaagttg aacgagaaac gtaaaatgat
     8401 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
     8461 acacaacata tccagtcact atgccatcca gctgatatcc cctatagtga gtcgtattac
     8521 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     8581 gcacaagata aaaatatatc atcatgcctc ctc
//
