LOCUS       sequence-350122-        7662 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 7662)
  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 7662)
  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-L2-dMb2Cas12a
FEATURES             Location/Qualifiers
     source          1..7662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      616..1204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1105..1124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1358..1375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1534..1561)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1653..1739)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1788..1810
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1802..1819
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1803..1819
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1835..1934
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1941..1961
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1968..2555
                     /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             2613..2633
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6408..6455
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6486..6738
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6783..6906
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6923..6942)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6924..6942)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6947..6963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6947..6963)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(7394..7662,1..523)
                     /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"
ORIGIN
        1 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
       61 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
      121 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
      181 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
      241 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
      301 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
      361 ctgggcaatg gagcgcctgc cggcccggta tcagcccgtc atacttgaag ctagacaggc
      421 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt
      481 ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga gccacccatg
      541 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
      601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
      661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
      781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1261 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1321 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1381 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1441 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg
     1501 taccgcgagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc
     1561 tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
     1621 tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc
     1681 gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga
     1741 tgcctggcag ttccctactc tcgcgttaac gcttgcatgg atgttttccc agtcacgacg
     1801 ttgtaaaacg acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat
     1861 agtgacctgt tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta
     1921 caaaaaagca ggctccgatg cccaaaaaaa aaagaaaggt ctcgtcacag ctcgtcaaga
     1981 gcgaactgga agagaagaaa tcggaattgc gccacaaact caaatacgtc cctcatgagt
     2041 acatcgaact catagaaatc gccagaaatt ctacccaaga ccggatactt gaaatgaagg
     2101 tcatggaatt ttttatgaag gtgtatgggt atagaggaaa gcacctcggt ggcagtagga
     2161 agcccgatgg cgcgatctac accgtcggta gtccgattga ctatggagtg atagtcgaca
     2221 caaaagcgta tagtggaggg tataacctgc ccattggaca agcagatgaa atgcagcggt
     2281 acgtggaaga aaaccaaaca cgcaataaac atataaatcc taacgaatgg tggaaggttt
     2341 atccctcctc cgttacagaa ttcaagtttt tgttcgtctc gggccacttc aagggcaact
     2401 acaaggccca gcttactcgg ttgaatcaca tcacaaattg caatggggca gtcttgagtg
     2461 tggaggaact tttgatcggg ggtgaaatga tcaaggccgg tacgctgacg ttggaggagg
     2521 ttcggcggaa gtttaataat ggcgaaataa acttttcagg gagtgagacg ccaggaacat
     2581 cagaaagcgc aaccccggag tcgaccatgg ctcctaagaa gaagcggaag gttggtattc
     2641 acggggtgcc tgcggctctg tttcaagatt ttacacatct gtacccgctg agtaaaacag
     2701 tgcggttcga gctgaaaccc ataggaagga ccctcgagca catccacgcg aagaattttc
     2761 tgagccagga tgaaactatg gctgatatgt atcaaaaagt taaggtcatt ttggacgact
     2821 atcatcgcga ttttattgcc gacatgatgg gagaggtgaa actcacgaag cttgctgaat
     2881 tttacgacgt ctatctgaag ttcaggaaaa atcctaagga cgatgggctg caaaaacagc
     2941 ttaaagacct tcaagctgtc cttcggaagg aatcggtgaa gcctataggg tcaggtggga
     3001 agtacaaaac aggctacgat agactctttg gggcaaaact cttcaaagat ggaaaagagt
     3061 tgggtgacct cgcaaaattc gttatagccc aagaaggtga gtcttctccg aagctggctc
     3121 atcttgctca ttttgagaag ttcagcacgt attttactgg atttcacgat aatcggaaga
     3181 atatgtactc ggatgaagac aagcatactg caatagcgta caggctcatc catgagaatt
     3241 tgccgagatt catcgacaat ctgcaaatct tgacaacaat caaacaaaag catagcgccc
     3301 tctatgatca gataatcaac gagctcacgg cctccgggct cgacgtctcc ttggcttctc
     3361 atcttgacgg gtatcacaag ctccttacac aagaggggat cacggcatac aacaggatca
     3421 taggagaggt gaatggatat acaaataagc ataaccagat atgccacaag agcgagcgca
     3481 tagcgaaact tagacccttg cacaagcaaa tcctttctga cggaatggga gtgtcattcc
     3541 ttccgtctaa gttcgcggat gatagtgaga tgtgccaagc ggtcaacgaa ttttatcgcc
     3601 attatactga cgtgttcgca aaggtgcaaa gtctctttga cggatttgat gatcaccaga
     3661 aagacgggat ctatgttgaa cacaaaaacc ttaatgaact gagcaaacag gcgttcggcg
     3721 actttgcttt gctggggagg gtccttgatg gatactacgt ggacgttgtc aatccggagt
     3781 tcaatgagcg gttcgcaaag gccaagactg acaatgcgaa agccaagctt acaaaagaaa
     3841 aggacaaatt cattaaagga gtccactcac tggcttccct cgaacaagca atagaacacc
     3901 atacagctag acacgacgat gagagtgttc aagccggaaa acttggccag tacttcaaac
     3961 acggtttggc gggggttgac aacccgattc agaaaattca caataaccat tcgacgatta
     4021 aagggtttct ggaaagggaa aggcctgctg gggaacgggc gctcccgaag atcaagtcag
     4081 gaaaaaaccc agaaatgaca cagctcaggc agctgaagga acttttggac aacgcattga
     4141 atgtggcgca cttcgctaag ctgctgacaa ctaaaacaac cttggacaac caggatggaa
     4201 atttttacgg ggagtttggg gtgctttacg acgagctggc taaaattcca actctctaca
     4261 ataaggttag agattatctc tctcaaaagc ccttttctac cgaaaagtat aagctcaact
     4321 tcggcaatcc gacccttctc aatgggtggg acctgaacaa agagaaagat aactttgggg
     4381 ttatacttca gaaggatgga tgctattact tggcgcttct tgataaggct cataaaaaag
     4441 ttttcgacaa cgcccctaac actggtaaga acgtctacca aaagatggtc tacaaactgt
     4501 tgcccggccc caacaaaatg cttcctaaag tgtttttcgc aaaatcgaat ctcgactatt
     4561 ataatccatc tgccgagctc cttgacaaat atgctaaggg gacccataaa aagggtgata
     4621 atttcaacct gaaggactgc cacgcgctta tcgacttttt caaagccggg ataaataagc
     4681 atccggagtg gcaacatttt ggttttaaat tttcgccaac gtcgtcctat cgcgaccttt
     4741 ccgatttcta tagggaagtt gaacctcagg ggtaccaggt caaatttgtt gacattaatg
     4801 cggactacat tgatgaattg gtggagcaag ggaagctcta cctctttcaa atatataaca
     4861 aagatttctc gccaaaagcg catggtaaac cgaatcttca taccttgtac tttaaagcac
     4921 ttttttcaga agataacttg gcggacccga tctacaagct gaatggggaa gctcagatct
     4981 tctacaggaa agcttcgttg gacatgaacg agactaccat acatcgcgcg ggagaggtgc
     5041 ttgagaacaa aaatcccgac aacccgaaaa agcggcaatt cgtttacgac atcatcaaag
     5101 acaaacggta cacgcaggac aaatttatgc tccacgtccc cattaccatg aattttggag
     5161 tccaaggcat gaccattaag gaattcaaca aaaaggtcaa ccaaagtatt cagcaatacg
     5221 atgaagtcaa tgtcataggc atagctcggg gagaaaggca tctgttgtat cttaccgtga
     5281 ttaactctaa gggtgaaata ctggagcaac ggtcacttaa cgatataacc acggcgtccg
     5341 cgaacggtac acaagtgacc actccctacc acaaaatatt ggataaaagg gagatagaac
     5401 gcttgaatgc ccgcgttggc tggggtgaga ttgagaccat caaagagctt aaatcgggat
     5461 atttgtctca cgtcgttcat caaattaacc aactcatgct taagtacaat gcaatcgttg
     5521 tgctcgagga cctgaacttt ggtttcaaaa gagggaggtt caaggtggaa aaacaaattt
     5581 accagaactt tgaaaacgcg cttatcaaga aattgaatca ccttgttttg aaagataagg
     5641 cagatgacga aatcgggtcg tataaaaatg cactccagtt gacaaataat ttcacggatt
     5701 tgaagtcgat cggcaagcaa acagggttcc tcttttatgt gccagcgtgg aatacatcaa
     5761 aaattgatcc ggagacggga tttgtcgact tgctgaagcc taggtatgag aacattgccc
     5821 aatctcaggc ctttttcggc aaattcgata aaatatgcta caacacagac aaaggttatt
     5881 ttgaatttca cattgattac gccaaattta cagataaggc gaaaaacagc agacagaaat
     5941 gggctatctg ttctcatggg gacaaacgct atgtctacga taagacggct aatcaaaata
     6001 aaggcgccgc aaaaggtatt aatgtgaatg atgagctgaa aagcttgttt gcccgctacc
     6061 atatcaatga taaacaacca aacttggtga tggacatatg ccagaacaat gacaaagaat
     6121 tccacaagtc actcatgtgc ctgcttaaaa cccttttggc gctgcggtat agcaatgcat
     6181 ctagcgatga agactttatt ttgagtcccg tggccaacga cgagggcgtg ttttttaatt
     6241 cagccttggc ggacgatacg cagccccaga atgcggacgc aaacggcgcg taccacattg
     6301 cactgaaggg actgtggctt ctgaacgagc tgaaaaatag cgacgacctg aataaagtca
     6361 agttggccat tgacaatcaa acctggttga atttcgctca aaatagaaag cgtcctgctg
     6421 ccaccaaaaa ggccggacag gctaagaaaa agaagtgaga cgactagtgg cggccgccga
     6481 cgtccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc
     6541 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt
     6601 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt
     6661 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt
     6721 catctatgtt actagatcgg gaattgatcc cccctcgaca gcttccggaa agggcgaatt
     6781 cgcaactttg tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt
     6841 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     6901 actatgccat ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     6961 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     7021 atcatcatgc ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa
     7081 ggttgccggg tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc
     7141 ctgttcggtt cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac
     7201 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
     7261 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
     7321 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
     7381 aaagttaaac attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
     7441 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
     7501 cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt
     7561 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
     7621 ccctggagag agcgagattc tccgcgctgt agaagtcacc at
//
