LOCUS       sequence-350120-        8429 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 8429)
  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 8429)
  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-NF1-L2-dSpRY
FEATURES             Location/Qualifiers
     source          1..8429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             686..694
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             774..821
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    1496..1619
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1636..1655)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1637..1655)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1660..1676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1660..1676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2107..2898
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2991..3579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3480..3499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3733..3750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3909..3936)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4028..4114)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4163..4185
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4177..4194
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4178..4194
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4210..4309
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4316..4336
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4343..4930
                     /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             4985..5050
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5057..5077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 ggcctaagcg gaacagcgac aagctcatcg cccgcaagaa ggactgggat ccgaagaagt
       61 acggcgggtt cctgtggccc actgtggcct actcggtcct ggttgtggcg aaggttgaga
      121 agggcaagtc caagaagctc aagagcgtga aggagctgct ggggatcacg attatggagc
      181 gctccagctt cgagaagaac ccgatcgatt tcctggaggc gaagggctac aaggaggtga
      241 agaaggacct gatcattaag ctccccaagt actcactctt cgagctggag aacggcagga
      301 agcggatgct ggcttccgct aagcagctgc agaaggggaa cgagctggct ctgccgtcca
      361 agtatgtgaa cttcctctac ctggcctccc actacgagaa gctcaagggc agccccgagg
      421 acaacgagca gaagcagctg ttcgtcgagc agcacaagca ttacctcgac gagatcattg
      481 agcagatttc cgagttctcc aagcgcgtga tcctggccga cgcgaatctg gataaggtcc
      541 tctccgcgta caacaagcac cgcgacaagc caatcaggga gcaggctgag aatatcattc
      601 atctcttcac cctgacgagg ctcggcgccc ctagggcttt caagtacttc gacacaacta
      661 tcgatccgaa gcagtacagg agcactaagg aggtcctgga cgcgaccctc atccaccagt
      721 cgattaccgg cctctacgag acgcgcatcg acctgtctca gctcgggggc gacaagcggc
      781 cagcggcgac gaagaaggcg gggcaggcga agaagaagaa gtgagctcag agctttcgtt
      841 cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat tgcgcacaca
      901 ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc ttgtaccatt
      961 tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac tgtgaaatgg
     1021 aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc aaattaatat
     1081 tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag atatgcaaac
     1141 attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag ttaatatgag
     1201 gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa tatattttca
     1261 gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt tttagacatt
     1321 tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc attgctttat
     1381 aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta atgcatttta
     1441 tgacttgcca attgttccgg aactctagat aagcttaccg gaaagggcga attcgcaact
     1501 ttgtatacaa aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     1561 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgc
     1621 catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     1681 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     1741 tgcctcctct ggaccagcca ggacagaaat gcctcgactt cgctgctacc caaggttgcc
     1801 gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg
     1861 gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc
     1921 gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt
     1981 ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt
     2041 ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta
     2101 aacattatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc
     2161 gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg
     2221 gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt
     2281 gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga
     2341 gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg
     2401 tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt
     2461 gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca
     2521 agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct
     2581 gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac
     2641 tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta
     2701 accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc
     2761 cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc
     2821 ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc
     2881 aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa cgtgagttac
     2941 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3001 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3061 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3121 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3181 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3241 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3301 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3361 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3421 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3481 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3541 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3601 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3661 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3721 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     3781 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3841 aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg cgagccagga agagtttgta
     3901 gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt
     3961 atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc
     4021 ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
     4081 cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgcg
     4141 ttaacgcttg catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaag
     4201 ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt
     4261 gatgagcaat gcttttttat aatgccaact ttgtacaaaa aagcaggctc cgatgccaaa
     4321 aaaaaagcgg aaggttagtt cacaactggt gaagtctgaa cttgaggaga agaagagcga
     4381 gttgaggcac aagttgaagt atgtccccca tgaatacatc gagttgattg agattgctcg
     4441 caacagcacc caggaccgga ttctcgaaat gaaggtcatg gaatttttta tgaaagtgta
     4501 cgggtaccgc ggaaagcatc tcgggggaag tcgcaaacct gacggggcta tctatacggt
     4561 gggttctcct atagactacg gggtcattgt ggatacgaag gcctatagtg gcggatacaa
     4621 tctgccaatt ggacaagctg atgaaatgca gaggtacgtt gaagagaatc agacccgcaa
     4681 caaacatata aacccaaatg aatggtggaa ggtgtacccc agcagtgtca ccgaattcaa
     4741 atttctgttt gtgagtgggc atttcaaagg aaactataaa gcacaactta cgagactgaa
     4801 ccatattacc aattgtaacg gcgctgtgtt gagtgtcgag gagctgctca tagggggaga
     4861 aatgatcaaa gcaggcacat tgactctcga ggaggtgcgg cggaagttta acaatgggga
     4921 aatcaacttc tcagggagtg agacgccagg aacatcagaa agcgcaaccc cggagtcgac
     4981 catggattac aaggaccacg acggggatta caaggaccac gacattgatt acaaggatga
     5041 tgatgacaag atggctccga agaagaagag gaaggttggc atccacgggg tgccagctgc
     5101 tgacaagaag tactcgatcg gcctcgccat tgggactaac tctgttggct gggccgtgat
     5161 caccgacgag tacaaggtgc cctcaaagaa gttcaaggtc ctgggcaaca ccgatcggca
     5221 ttccatcaag aagaatctca ttggcgctct cctgttcgac agcggcgaga cggctgagag
     5281 gacgcggctc aagcgcaccg cccgcaggcg gtacacgcgc aggaagaatc gcatctgcta
     5341 cctgcaggag attttctcca acgagatggc gaaggttgac gattctttct tccacaggct
     5401 ggaggagtca ttcctcgtgg aggaggataa gaagcacgag cggcatccaa tcttcggcaa
     5461 cattgtcgac gaggttgcct accacgagaa gtaccctacg atctaccatc tgcggaagaa
     5521 gctcgtggac tccacagata aggcggacct ccgcctgatc tacctcgctc tggcccacat
     5581 gattaagttc aggggccatt tcctgatcga gggggatctc aacccggaca atagcgatgt
     5641 tgacaagctg ttcatccagc tcgtgcagac gtacaaccag ctcttcgagg agaaccccat
     5701 taatgcgtca ggcgtcgacg cgaaggctat cctgtccgct aggctctcga agtctcggcg
     5761 cctcgagaac ctgatcgccc agctgccggg cgagaagaag aacggcctgt tcgggaatct
     5821 cattgcgctc agcctggggc tcacgcccaa cttcaagtcg aatttcgatc tcgctgagga
     5881 cgccaagctg cagctctcca aggacacata cgacgatgac ctggataacc tcctggccca
     5941 gatcggcgat cagtacgcgg acctgttcct cgctgccaag aatctgtcgg acgccatcct
     6001 cctgtctgat attctcaggg tgaacaccga gattacgaag gctccgctct cagcctccat
     6061 gatcaagcgc tacgacgagc accatcagga tctgaccctc ctgaaggcgc tggtcaggca
     6121 gcagctcccc gagaagtaca aggagatctt cttcgatcag tcgaagaacg gctacgctgg
     6181 gtacattgac ggcggggcct ctcaggagga gttctacaag ttcatcaagc cgattctgga
     6241 gaagatggac ggcacggagg agctgctggt gaagctcaat cgcgaggacc tcctgaggaa
     6301 gcagcggaca ttcgataacg gcagcatccc acaccagatt catctcgggg agctgcacgc
     6361 tatcctgagg aggcaggagg acttctaccc tttcctcaag gataaccgcg agaagatcga
     6421 gaagattctg actttcagga tcccgtacta cgtcggccca ctcgctaggg gcaactcccg
     6481 cttcgcttgg atgacccgca agtcagagga gacgatcacg ccgtggaact tcgaggaggt
     6541 ggtcgacaag ggcgctagcg ctcagtcgtt catcgagagg atgacgaatt tcgacaagaa
     6601 cctgccaaat gagaaggtgc tccctaagca ctcgctcctg tacgagtact tcacagtcta
     6661 caacgagctg actaaggtga agtatgtgac cgagggcatg aggaagccgg ctttcctgtc
     6721 tggggagcag aagaaggcca tcgtggacct cctgttcaag accaaccgga aggtcacggt
     6781 taagcagctc aaggaggact acttcaagaa gattgagtgc ttcgattcgg tcgagatctc
     6841 tggcgttgag gaccgcttca acgcctccct ggggacctac cacgatctcc tgaagatcat
     6901 taaggataag gacttcctgg acaacgagga gaatgaggat atcctcgagg acattgtgct
     6961 gacactcact ctgttcgagg accgggagat gatcgaggag cgcctgaaga cttacgccca
     7021 tctcttcgat gacaaggtca tgaagcagct caagaggagg aggtacaccg gctgggggag
     7081 gctgagcagg aagctcatca acggcattcg ggacaagcag tccgggaaga cgatcctcga
     7141 cttcctgaag agcgatggct tcgcgaaccg caatttcatg cagctgattc acgatgacag
     7201 cctcacattc aaggaggata tccagaaggc tcaggtgagc ggccaggggg actcgctgca
     7261 cgagcatatc gcgaacctcg ctggctcgcc agctatcaag aaggggattc tgcagaccgt
     7321 gaaggttgtg gacgagctgg tgaaggtcat gggcaggcac aagcctgaga acatcgtcat
     7381 tgagatggcc cgggagaatc agaccacgca gaagggccag aagaactcac gcgagaggat
     7441 gaagaggatc gaggagggca ttaaggagct ggggtcccag atcctcaagg agcacccggt
     7501 ggagaacacg cagctgcaga atgagaagct ctacctgtac tacctccaga atggccgcga
     7561 tatgtatgtg gaccaggagc tggatattaa caggctcagc gattacgacg tcgatgctat
     7621 cgttccacag tcattcctga aggatgactc cattgacaac aaggtcctca ccaggtcgga
     7681 caagaaccgg ggcaagtctg ataatgttcc ttcagaggag gtcgttaaga agatgaagaa
     7741 ctactggcgc cagctcctga atgccaagct gatcacgcag cggaagttcg ataacctcac
     7801 aaaggctgag aggggcgggc tctctgagct ggacaaggcg ggcttcatca agaggcagct
     7861 ggtcgagaca cggcagatca ctaagcacgt tgcgcagatt ctcgactcac ggatgaacac
     7921 taagtacgat gagaatgaca agctgatccg cgaggtgaag gtcatcaccc tgaagtcaaa
     7981 gctcgtctcc gacttcagga aggatttcca gttctacaag gttcgggaga tcaacaatta
     8041 ccaccatgcc catgacgcgt acctgaacgc ggtggtcggc acagctctga tcaagaagta
     8101 cccaaagctc gagagcgagt tcgtgtacgg ggactacaag gtttacgatg tgaggaagat
     8161 gatcgccaag tcggagcagg agattggcaa ggctaccgcc aagtacttct tctactctaa
     8221 cattatgaat ttcttcaaga cagagatcac tctggccaat ggcgagatcc ggaagcgccc
     8281 cctcatcgag acgaacggcg agacggggga gatcgtgtgg gacaagggca gggatttcgc
     8341 gaccgtcagg aaggttctct ccatgccaca agtgaatatc gtcaagaaga cagaggtcca
     8401 gactggcggg ttctctaagg agtcaatta
//
