LOCUS       sequence-350121-        7689 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 7689)
  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 7689)
  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-L1-dMb2Cas12a
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             430..1221
                     /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      1314..1902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1803..1822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2056..2073
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2232..2259)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2351..2437)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2486..2508
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2500..2517
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2501..2517
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2533..2632
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2639..2659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2666..3253
                     /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             3338..3358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7133..7180
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7211..7463
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7508..7631
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7648..7667)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7649..7667)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7672..7688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7672..7688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
       61 tcatgcctcc tctggaccag ccaggacaga aatgcctcga cttcgctgct acccaaggtt
      121 gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa cccagtggac ataagcctgt
      181 tcggttcgta agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg
      241 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt
      301 tttggggtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga
      361 tgtttgatgt tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag
      421 ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt
      481 ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca
      541 gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg
      601 cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct
      661 ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt
      721 ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt
      781 cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa
      841 gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt
      901 cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc
      961 gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca
     1021 gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg
     1081 gcccggtatc agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat
     1141 cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc
     1201 accaaggtag tcggcaaata accctcgagc cacccatgac caaaatccct taacgtgagt
     1261 tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1321 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1381 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1441 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     1501 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1561 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1621 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1681 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1741 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1801 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1861 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     1921 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1981 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2041 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     2101 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2161 tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca ggaagagttt
     2221 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
     2281 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
     2341 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
     2401 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     2461 gcgttaacgc ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt
     2521 aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
     2581 attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg ctccgatgcc
     2641 aaaaaaaaag cggaaggtta gttcacaact ggtgaagtct gaacttgagg agaagaagag
     2701 cgagttgagg cacaagttga agtatgtccc ccatgaatac atcgagttga ttgagattgc
     2761 tcgcaacagc acccaggacc ggattctcga aatgaaggtc atggaatttt ttatgaaagt
     2821 gtacgggtac cgcggaaagc atctcggggg aagtcgcaaa cctgacgggg ctatctatac
     2881 ggtgggttct cctatagact acggggtcat tgtggatacg aaggcctata gtggcggata
     2941 caatctgcca attggacaag ctgatgaaat gcagaggtac gttgaagaga atcagacccg
     3001 caacaaacat ataaacccaa atgaatggtg gaaggtgtac cccagcagtg tcaccgaatt
     3061 caaatttctg tttgtgagtg ggcatttcaa aggaaactat aaagcacaac ttacgagact
     3121 gaaccatatt accaattgta acggcgctgt gttgagtgtc gaggagctgc tcataggggg
     3181 agaaatgatc aaagcaggca cattgactct cgaggaggtg cggcggaagt ttaacaatgg
     3241 ggaaatcaac ttcggtggtg gtggttctgg aggtggaggc tctggaggag gaggcagcgg
     3301 tggaggcggg tctggaggtg gtggttctac catggctcct aagaagaagc ggaaggttgg
     3361 tattcacggg gtgcctgcgg ctctgtttca agattttaca catctgtacc cgctgagtaa
     3421 aacagtgcgg ttcgagctga aacccatagg aaggaccctc gagcacatcc acgcgaagaa
     3481 ttttctgagc caggatgaaa ctatggctga tatgtatcaa aaagttaagg tcattttgga
     3541 cgactatcat cgcgatttta ttgccgacat gatgggagag gtgaaactca cgaagcttgc
     3601 tgaattttac gacgtctatc tgaagttcag gaaaaatcct aaggacgatg ggctgcaaaa
     3661 acagcttaaa gaccttcaag ctgtccttcg gaaggaatcg gtgaagccta tagggtcagg
     3721 tgggaagtac aaaacaggct acgatagact ctttggggca aaactcttca aagatggaaa
     3781 agagttgggt gacctcgcaa aattcgttat agcccaagaa ggtgagtctt ctccgaagct
     3841 ggctcatctt gctcattttg agaagttcag cacgtatttt actggatttc acgataatcg
     3901 gaagaatatg tactcggatg aagacaagca tactgcaata gcgtacaggc tcatccatga
     3961 gaatttgccg agattcatcg acaatctgca aatcttgaca acaatcaaac aaaagcatag
     4021 cgccctctat gatcagataa tcaacgagct cacggcctcc gggctcgacg tctccttggc
     4081 ttctcatctt gacgggtatc acaagctcct tacacaagag gggatcacgg catacaacag
     4141 gatcatagga gaggtgaatg gatatacaaa taagcataac cagatatgcc acaagagcga
     4201 gcgcatagcg aaacttagac ccttgcacaa gcaaatcctt tctgacggaa tgggagtgtc
     4261 attccttccg tctaagttcg cggatgatag tgagatgtgc caagcggtca acgaatttta
     4321 tcgccattat actgacgtgt tcgcaaaggt gcaaagtctc tttgacggat ttgatgatca
     4381 ccagaaagac gggatctatg ttgaacacaa aaaccttaat gaactgagca aacaggcgtt
     4441 cggcgacttt gctttgctgg ggagggtcct tgatggatac tacgtggacg ttgtcaatcc
     4501 ggagttcaat gagcggttcg caaaggccaa gactgacaat gcgaaagcca agcttacaaa
     4561 agaaaaggac aaattcatta aaggagtcca ctcactggct tccctcgaac aagcaataga
     4621 acaccataca gctagacacg acgatgagag tgttcaagcc ggaaaacttg gccagtactt
     4681 caaacacggt ttggcggggg ttgacaaccc gattcagaaa attcacaata accattcgac
     4741 gattaaaggg tttctggaaa gggaaaggcc tgctggggaa cgggcgctcc cgaagatcaa
     4801 gtcaggaaaa aacccagaaa tgacacagct caggcagctg aaggaacttt tggacaacgc
     4861 attgaatgtg gcgcacttcg ctaagctgct gacaactaaa acaaccttgg acaaccagga
     4921 tggaaatttt tacggggagt ttggggtgct ttacgacgag ctggctaaaa ttccaactct
     4981 ctacaataag gttagagatt atctctctca aaagcccttt tctaccgaaa agtataagct
     5041 caacttcggc aatccgaccc ttctcaatgg gtgggacctg aacaaagaga aagataactt
     5101 tggggttata cttcagaagg atggatgcta ttacttggcg cttcttgata aggctcataa
     5161 aaaagttttc gacaacgccc ctaacactgg taagaacgtc taccaaaaga tggtctacaa
     5221 actgttgccc ggccccaaca aaatgcttcc taaagtgttt ttcgcaaaat cgaatctcga
     5281 ctattataat ccatctgccg agctccttga caaatatgct aaggggaccc ataaaaaggg
     5341 tgataatttc aacctgaagg actgccacgc gcttatcgac tttttcaaag ccgggataaa
     5401 taagcatccg gagtggcaac attttggttt taaattttcg ccaacgtcgt cctatcgcga
     5461 cctttccgat ttctataggg aagttgaacc tcaggggtac caggtcaaat ttgttgacat
     5521 taatgcggac tacattgatg aattggtgga gcaagggaag ctctacctct ttcaaatata
     5581 taacaaagat ttctcgccaa aagcgcatgg taaaccgaat cttcatacct tgtactttaa
     5641 agcacttttt tcagaagata acttggcgga cccgatctac aagctgaatg gggaagctca
     5701 gatcttctac aggaaagctt cgttggacat gaacgagact accatacatc gcgcgggaga
     5761 ggtgcttgag aacaaaaatc ccgacaaccc gaaaaagcgg caattcgttt acgacatcat
     5821 caaagacaaa cggtacacgc aggacaaatt tatgctccac gtccccatta ccatgaattt
     5881 tggagtccaa ggcatgacca ttaaggaatt caacaaaaag gtcaaccaaa gtattcagca
     5941 atacgatgaa gtcaatgtca taggcatagc tcggggagaa aggcatctgt tgtatcttac
     6001 cgtgattaac tctaagggtg aaatactgga gcaacggtca cttaacgata taaccacggc
     6061 gtccgcgaac ggtacacaag tgaccactcc ctaccacaaa atattggata aaagggagat
     6121 agaacgcttg aatgcccgcg ttggctgggg tgagattgag accatcaaag agcttaaatc
     6181 gggatatttg tctcacgtcg ttcatcaaat taaccaactc atgcttaagt acaatgcaat
     6241 cgttgtgctc gaggacctga actttggttt caaaagaggg aggttcaagg tggaaaaaca
     6301 aatttaccag aactttgaaa acgcgcttat caagaaattg aatcaccttg ttttgaaaga
     6361 taaggcagat gacgaaatcg ggtcgtataa aaatgcactc cagttgacaa ataatttcac
     6421 ggatttgaag tcgatcggca agcaaacagg gttcctcttt tatgtgccag cgtggaatac
     6481 atcaaaaatt gatccggaga cgggatttgt cgacttgctg aagcctaggt atgagaacat
     6541 tgcccaatct caggcctttt tcggcaaatt cgataaaata tgctacaaca cagacaaagg
     6601 ttattttgaa tttcacattg attacgccaa atttacagat aaggcgaaaa acagcagaca
     6661 gaaatgggct atctgttctc atggggacaa acgctatgtc tacgataaga cggctaatca
     6721 aaataaaggc gccgcaaaag gtattaatgt gaatgatgag ctgaaaagct tgtttgcccg
     6781 ctaccatatc aatgataaac aaccaaactt ggtgatggac atatgccaga acaatgacaa
     6841 agaattccac aagtcactca tgtgcctgct taaaaccctt ttggcgctgc ggtatagcaa
     6901 tgcatctagc gatgaagact ttattttgag tcccgtggcc aacgacgagg gcgtgttttt
     6961 taattcagcc ttggcggacg atacgcagcc ccagaatgcg gacgcaaacg gcgcgtacca
     7021 cattgcactg aagggactgt ggcttctgaa cgagctgaaa aatagcgacg acctgaataa
     7081 agtcaagttg gccattgaca atcaaacctg gttgaatttc gctcaaaata gaaagcgtcc
     7141 tgctgccacc aaaaaggccg gacaggctaa gaaaaagaag tgagacgact agtggcggcc
     7201 gccgacgtcc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc
     7261 cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa
     7321 catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata
     7381 catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
     7441 ggtgtcatct atgttactag atcgggaatt gatcccccct cgacagcttc cggaaagggc
     7501 gaattcgcaa ctttgtatac aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat
     7561 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
     7621 cagtcactat gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
     7681 gtttcctgg
//
