LOCUS       sequence-350123-        8613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8613)
  AUTHORS   Cheng Y, Sretenovic S, Zhang Y, Pan C, Huang J, Qi Y
  TITLE     Expanding the targeting scope of FokI-dCas nuclease systems with 
            SpRY and Mb2Cas12a.
  JOURNAL   Biotechnol J. 2022 Apr 4:e2100571. doi: 10.1002/biot.202100571.
  PUBMED    35377968
REFERENCE   2  (bases 1 to 8613)
  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"; journalName: "Biotechnol 
            J. 2022 Apr 4:e2100571. doi: 10.1002/biot.202100571."
COMMENT     Sequence Label: pYPQ-NF-L3-NF-L1-dMb2Cas12a
FEATURES             Location/Qualifiers
     source          1..8613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             125..916
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPARYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1009..1597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1498..1517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1751..1768
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1927..1954)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2046..2132)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2181..2203
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2195..2212
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2196..2212
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2228..2327
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2334..2354
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2361..2948
                     /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"
     CDS             3258..3278
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3285..3872
                     /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"
     CDS             3957..3977
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7752..7799
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7830..8082
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    8127..8250
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8267..8286)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8268..8286)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8291..8307)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8291..8307)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
       61 gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa
      121 cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt
      181 catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga
      241 tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga
      301 tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga
      361 gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg
      421 gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc
      481 aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag
      541 agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga
      601 acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg
      661 ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac
      721 cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggcccg
      781 gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt
      841 ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa
      901 ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc
      961 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1021 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1081 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1141 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1201 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1261 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1321 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1381 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1441 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1501 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1561 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1621 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     1681 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     1741 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     1801 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1861 agcgggcagt gagcgcaacg caattaatac gcgtaccgcg agccaggaag agtttgtaga
     1921 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
     1981 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
     2041 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     2101 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
     2161 aacgcttgca tggatgtttt cccagtcacg acgttgtaaa acgacggcca gtcttaagct
     2221 cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
     2281 tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggctccg atgcccaaaa
     2341 aaaaaagaaa ggtctcgtca cagctcgtca agagcgaact ggaagagaag aaatcggaat
     2401 tgcgccacaa actcaaatac gtccctcatg agtacatcga actcatagaa atcgccagaa
     2461 attctaccca agaccggata cttgaaatga aggtcatgga attttttatg aaggtgtatg
     2521 ggtatagagg aaagcacctc ggtggcagta ggaagcccga tggcgcgatc tacaccgtcg
     2581 gtagtccgat tgactatgga gtgatagtcg acacaaaagc gtatagtgga gggtataacc
     2641 tgcccattgg acaagcagat gaaatgcagc ggtacgtgga agaaaaccaa acacgcaata
     2701 aacatataaa tcctaacgaa tggtggaagg tttatccctc ctccgttaca gaattcaagt
     2761 ttttgttcgt ctcgggccac ttcaagggca actacaaggc ccagcttact cggttgaatc
     2821 acatcacaaa ttgcaatggg gcagtcttga gtgtggagga acttttgatc gggggtgaaa
     2881 tgatcaaggc cggtacgctg acgttggagg aggttcggcg gaagtttaat aatggcgaaa
     2941 taaactttac cggtgctagc gggtctgggt caggttccgg ctcgattact cggacaacaa
     3001 atccacggaa cgtcgttcct aaaatctata tgtcagcagg gtcgatacca ctgacgacgc
     3061 atataaccaa tagcattcag cctaccctgt ggacgattgg gtccatcaac ggtgtcgccc
     3121 ccttggcgaa gtctataaaa cttgggatac ccgtcactgg atcagcatac accgaccaga
     3181 cgactgctat ggttaggaag aaagtgtctg tttttatggg aagtggttca ggatccggct
     3241 caagtctgca gactagtcca aaaaaaaagc ggaaggttag ttcacaactg gtgaagtctg
     3301 aacttgagga gaagaagagc gagttgaggc acaagttgaa gtatgtcccc catgaataca
     3361 tcgagttgat tgagattgct cgcaacagca cccaggaccg gattctcgaa atgaaggtca
     3421 tggaattttt tatgaaagtg tacgggtacc gcggaaagca tctcggggga agtcgcaaac
     3481 ctgacggggc tatctatacg gtgggttctc ctatagacta cggggtcatt gtggatacga
     3541 aggcctatag tggcggatac aatctgccaa ttggacaagc tgatgaaatg cagaggtacg
     3601 ttgaagagaa tcagacccgc aacaaacata taaacccaaa tgaatggtgg aaggtgtacc
     3661 ccagcagtgt caccgaattc aaatttctgt ttgtgagtgg gcatttcaaa ggaaactata
     3721 aagcacaact tacgagactg aaccatatta ccaattgtaa cggcgctgtg ttgagtgtcg
     3781 aggagctgct cataggggga gaaatgatca aagcaggcac attgactctc gaggaggtgc
     3841 ggcggaagtt taacaatggg gaaatcaact tcggtggtgg tggttctgga ggtggaggct
     3901 ctggaggagg aggcagcggt ggaggcgggt ctggaggtgg tggttctacc atggctccta
     3961 agaagaagcg gaaggttggt attcacgggg tgcctgcggc tctgtttcaa gattttacac
     4021 atctgtaccc gctgagtaaa acagtgcggt tcgagctgaa acccatagga aggaccctcg
     4081 agcacatcca cgcgaagaat tttctgagcc aggatgaaac tatggctgat atgtatcaaa
     4141 aagttaaggt cattttggac gactatcatc gcgattttat tgccgacatg atgggagagg
     4201 tgaaactcac gaagcttgct gaattttacg acgtctatct gaagttcagg aaaaatccta
     4261 aggacgatgg gctgcaaaaa cagcttaaag accttcaagc tgtccttcgg aaggaatcgg
     4321 tgaagcctat agggtcaggt gggaagtaca aaacaggcta cgatagactc tttggggcaa
     4381 aactcttcaa agatggaaaa gagttgggtg acctcgcaaa attcgttata gcccaagaag
     4441 gtgagtcttc tccgaagctg gctcatcttg ctcattttga gaagttcagc acgtatttta
     4501 ctggatttca cgataatcgg aagaatatgt actcggatga agacaagcat actgcaatag
     4561 cgtacaggct catccatgag aatttgccga gattcatcga caatctgcaa atcttgacaa
     4621 caatcaaaca aaagcatagc gccctctatg atcagataat caacgagctc acggcctccg
     4681 ggctcgacgt ctccttggct tctcatcttg acgggtatca caagctcctt acacaagagg
     4741 ggatcacggc atacaacagg atcataggag aggtgaatgg atatacaaat aagcataacc
     4801 agatatgcca caagagcgag cgcatagcga aacttagacc cttgcacaag caaatccttt
     4861 ctgacggaat gggagtgtca ttccttccgt ctaagttcgc ggatgatagt gagatgtgcc
     4921 aagcggtcaa cgaattttat cgccattata ctgacgtgtt cgcaaaggtg caaagtctct
     4981 ttgacggatt tgatgatcac cagaaagacg ggatctatgt tgaacacaaa aaccttaatg
     5041 aactgagcaa acaggcgttc ggcgactttg ctttgctggg gagggtcctt gatggatact
     5101 acgtggacgt tgtcaatccg gagttcaatg agcggttcgc aaaggccaag actgacaatg
     5161 cgaaagccaa gcttacaaaa gaaaaggaca aattcattaa aggagtccac tcactggctt
     5221 ccctcgaaca agcaatagaa caccatacag ctagacacga cgatgagagt gttcaagccg
     5281 gaaaacttgg ccagtacttc aaacacggtt tggcgggggt tgacaacccg attcagaaaa
     5341 ttcacaataa ccattcgacg attaaagggt ttctggaaag ggaaaggcct gctggggaac
     5401 gggcgctccc gaagatcaag tcaggaaaaa acccagaaat gacacagctc aggcagctga
     5461 aggaactttt ggacaacgca ttgaatgtgg cgcacttcgc taagctgctg acaactaaaa
     5521 caaccttgga caaccaggat ggaaattttt acggggagtt tggggtgctt tacgacgagc
     5581 tggctaaaat tccaactctc tacaataagg ttagagatta tctctctcaa aagccctttt
     5641 ctaccgaaaa gtataagctc aacttcggca atccgaccct tctcaatggg tgggacctga
     5701 acaaagagaa agataacttt ggggttatac ttcagaagga tggatgctat tacttggcgc
     5761 ttcttgataa ggctcataaa aaagttttcg acaacgcccc taacactggt aagaacgtct
     5821 accaaaagat ggtctacaaa ctgttgcccg gccccaacaa aatgcttcct aaagtgtttt
     5881 tcgcaaaatc gaatctcgac tattataatc catctgccga gctccttgac aaatatgcta
     5941 aggggaccca taaaaagggt gataatttca acctgaagga ctgccacgcg cttatcgact
     6001 ttttcaaagc cgggataaat aagcatccgg agtggcaaca ttttggtttt aaattttcgc
     6061 caacgtcgtc ctatcgcgac ctttccgatt tctataggga agttgaacct caggggtacc
     6121 aggtcaaatt tgttgacatt aatgcggact acattgatga attggtggag caagggaagc
     6181 tctacctctt tcaaatatat aacaaagatt tctcgccaaa agcgcatggt aaaccgaatc
     6241 ttcatacctt gtactttaaa gcactttttt cagaagataa cttggcggac ccgatctaca
     6301 agctgaatgg ggaagctcag atcttctaca ggaaagcttc gttggacatg aacgagacta
     6361 ccatacatcg cgcgggagag gtgcttgaga acaaaaatcc cgacaacccg aaaaagcggc
     6421 aattcgttta cgacatcatc aaagacaaac ggtacacgca ggacaaattt atgctccacg
     6481 tccccattac catgaatttt ggagtccaag gcatgaccat taaggaattc aacaaaaagg
     6541 tcaaccaaag tattcagcaa tacgatgaag tcaatgtcat aggcatagct cggggagaaa
     6601 ggcatctgtt gtatcttacc gtgattaact ctaagggtga aatactggag caacggtcac
     6661 ttaacgatat aaccacggcg tccgcgaacg gtacacaagt gaccactccc taccacaaaa
     6721 tattggataa aagggagata gaacgcttga atgcccgcgt tggctggggt gagattgaga
     6781 ccatcaaaga gcttaaatcg ggatatttgt ctcacgtcgt tcatcaaatt aaccaactca
     6841 tgcttaagta caatgcaatc gttgtgctcg aggacctgaa ctttggtttc aaaagaggga
     6901 ggttcaaggt ggaaaaacaa atttaccaga actttgaaaa cgcgcttatc aagaaattga
     6961 atcaccttgt tttgaaagat aaggcagatg acgaaatcgg gtcgtataaa aatgcactcc
     7021 agttgacaaa taatttcacg gatttgaagt cgatcggcaa gcaaacaggg ttcctctttt
     7081 atgtgccagc gtggaataca tcaaaaattg atccggagac gggatttgtc gacttgctga
     7141 agcctaggta tgagaacatt gcccaatctc aggccttttt cggcaaattc gataaaatat
     7201 gctacaacac agacaaaggt tattttgaat ttcacattga ttacgccaaa tttacagata
     7261 aggcgaaaaa cagcagacag aaatgggcta tctgttctca tggggacaaa cgctatgtct
     7321 acgataagac ggctaatcaa aataaaggcg ccgcaaaagg tattaatgtg aatgatgagc
     7381 tgaaaagctt gtttgcccgc taccatatca atgataaaca accaaacttg gtgatggaca
     7441 tatgccagaa caatgacaaa gaattccaca agtcactcat gtgcctgctt aaaacccttt
     7501 tggcgctgcg gtatagcaat gcatctagcg atgaagactt tattttgagt cccgtggcca
     7561 acgacgaggg cgtgtttttt aattcagcct tggcggacga tacgcagccc cagaatgcgg
     7621 acgcaaacgg cgcgtaccac attgcactga agggactgtg gcttctgaac gagctgaaaa
     7681 atagcgacga cctgaataaa gtcaagttgg ccattgacaa tcaaacctgg ttgaatttcg
     7741 ctcaaaatag aaagcgtcct gctgccacca aaaaggccgg acaggctaag aaaaagaagt
     7801 gagacgacta gtggcggccg ccgacgtccg atcgttcaaa catttggcaa taaagtttct
     7861 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg
     7921 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     7981 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     8041 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaattg atcccccctc
     8101 gacagcttcc ggaaagggcg aattcgcaac tttgtataca aaagttgaac gagaaacgta
     8161 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
     8221 ctgtaaaaca caacatatcc agtcactatg ccatccagct gatatcccct atagtgagtc
     8281 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     8341 ttacattgca caagataaaa atatatcatc atgcctcctc tggaccagcc aggacagaaa
     8401 tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa
     8461 ggcacgaacc cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat
     8521 gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc
     8581 ggttttcatg gcttgttatg actgtttttt tgg
//
