LOCUS       sequence-350119-        8456 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway entry clone (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8456)
  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 8456)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-L1-dSpRY
FEATURES             Location/Qualifiers
     source          1..8456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             368..1159
                     /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      1252..1840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1741..1760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1994..2011
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2170..2197)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2289..2375)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2424..2446
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2438..2455
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2439..2455
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2471..2570
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2577..2597
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2604..3191
                     /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             3273..3338
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3345..3365
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             7403..7411
                     /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             7491..7538
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    8213..8336
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8353..8372)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8354..8372)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8377..8393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8377..8393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgcctcctc tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc
       61 cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc
      121 ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac
      181 cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt
      241 tggggtacag tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg
      301 tttgatgtta tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt
      361 aaacattatg agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg
      421 cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt
      481 ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct
      541 tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg
      601 agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc
      661 gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct
      721 tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc
      781 aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc
      841 tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga
      901 ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt
      961 aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc
     1021 ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg
     1081 cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac
     1141 caaggtagtc ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta
     1201 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1261 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1321 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1381 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1441 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1501 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1561 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1621 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1681 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1741 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1801 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1861 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1921 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1981 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     2041 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2101 gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt
     2161 agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt
     2221 tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct
     2281 cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag
     2341 gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
     2401 gttaacgctt gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa
     2461 gctcgggccc caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     2521 tgatgagcaa tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgatgccaa
     2581 aaaaaaagcg gaaggttagt tcacaactgg tgaagtctga acttgaggag aagaagagcg
     2641 agttgaggca caagttgaag tatgtccccc atgaatacat cgagttgatt gagattgctc
     2701 gcaacagcac ccaggaccgg attctcgaaa tgaaggtcat ggaatttttt atgaaagtgt
     2761 acgggtaccg cggaaagcat ctcgggggaa gtcgcaaacc tgacggggct atctatacgg
     2821 tgggttctcc tatagactac ggggtcattg tggatacgaa ggcctatagt ggcggataca
     2881 atctgccaat tggacaagct gatgaaatgc agaggtacgt tgaagagaat cagacccgca
     2941 acaaacatat aaacccaaat gaatggtgga aggtgtaccc cagcagtgtc accgaattca
     3001 aatttctgtt tgtgagtggg catttcaaag gaaactataa agcacaactt acgagactga
     3061 accatattac caattgtaac ggcgctgtgt tgagtgtcga ggagctgctc atagggggag
     3121 aaatgatcaa agcaggcaca ttgactctcg aggaggtgcg gcggaagttt aacaatgggg
     3181 aaatcaactt cggtggtggt ggttctggag gtggaggctc tggaggagga ggcagcggtg
     3241 gaggcgggtc tggaggtggt ggttctacca tggattacaa ggaccacgac ggggattaca
     3301 aggaccacga cattgattac aaggatgatg atgacaagat ggctccgaag aagaagagga
     3361 aggttggcat ccacggggtg ccagctgctg acaagaagta ctcgatcggc ctcgccattg
     3421 ggactaactc tgttggctgg gccgtgatca ccgacgagta caaggtgccc tcaaagaagt
     3481 tcaaggtcct gggcaacacc gatcggcatt ccatcaagaa gaatctcatt ggcgctctcc
     3541 tgttcgacag cggcgagacg gctgagagga cgcggctcaa gcgcaccgcc cgcaggcggt
     3601 acacgcgcag gaagaatcgc atctgctacc tgcaggagat tttctccaac gagatggcga
     3661 aggttgacga ttctttcttc cacaggctgg aggagtcatt cctcgtggag gaggataaga
     3721 agcacgagcg gcatccaatc ttcggcaaca ttgtcgacga ggttgcctac cacgagaagt
     3781 accctacgat ctaccatctg cggaagaagc tcgtggactc cacagataag gcggacctcc
     3841 gcctgatcta cctcgctctg gcccacatga ttaagttcag gggccatttc ctgatcgagg
     3901 gggatctcaa cccggacaat agcgatgttg acaagctgtt catccagctc gtgcagacgt
     3961 acaaccagct cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc
     4021 tgtccgctag gctctcgaag tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg
     4081 agaagaagaa cggcctgttc gggaatctca ttgcgctcag cctggggctc acgcccaact
     4141 tcaagtcgaa tttcgatctc gctgaggacg ccaagctgca gctctccaag gacacatacg
     4201 acgatgacct ggataacctc ctggcccaga tcggcgatca gtacgcggac ctgttcctcg
     4261 ctgccaagaa tctgtcggac gccatcctcc tgtctgatat tctcagggtg aacaccgaga
     4321 ttacgaaggc tccgctctca gcctccatga tcaagcgcta cgacgagcac catcaggatc
     4381 tgaccctcct gaaggcgctg gtcaggcagc agctccccga gaagtacaag gagatcttct
     4441 tcgatcagtc gaagaacggc tacgctgggt acattgacgg cggggcctct caggaggagt
     4501 tctacaagtt catcaagccg attctggaga agatggacgg cacggaggag ctgctggtga
     4561 agctcaatcg cgaggacctc ctgaggaagc agcggacatt cgataacggc agcatcccac
     4621 accagattca tctcggggag ctgcacgcta tcctgaggag gcaggaggac ttctaccctt
     4681 tcctcaagga taaccgcgag aagatcgaga agattctgac tttcaggatc ccgtactacg
     4741 tcggcccact cgctaggggc aactcccgct tcgcttggat gacccgcaag tcagaggaga
     4801 cgatcacgcc gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca
     4861 tcgagaggat gacgaatttc gacaagaacc tgccaaatga gaaggtgctc cctaagcact
     4921 cgctcctgta cgagtacttc acagtctaca acgagctgac taaggtgaag tatgtgaccg
     4981 agggcatgag gaagccggct ttcctgtctg gggagcagaa gaaggccatc gtggacctcc
     5041 tgttcaagac caaccggaag gtcacggtta agcagctcaa ggaggactac ttcaagaaga
     5101 ttgagtgctt cgattcggtc gagatctctg gcgttgagga ccgcttcaac gcctccctgg
     5161 ggacctacca cgatctcctg aagatcatta aggataagga cttcctggac aacgaggaga
     5221 atgaggatat cctcgaggac attgtgctga cactcactct gttcgaggac cgggagatga
     5281 tcgaggagcg cctgaagact tacgcccatc tcttcgatga caaggtcatg aagcagctca
     5341 agaggaggag gtacaccggc tgggggaggc tgagcaggaa gctcatcaac ggcattcggg
     5401 acaagcagtc cgggaagacg atcctcgact tcctgaagag cgatggcttc gcgaaccgca
     5461 atttcatgca gctgattcac gatgacagcc tcacattcaa ggaggatatc cagaaggctc
     5521 aggtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcgccag
     5581 ctatcaagaa ggggattctg cagaccgtga aggttgtgga cgagctggtg aaggtcatgg
     5641 gcaggcacaa gcctgagaac atcgtcattg agatggcccg ggagaatcag accacgcaga
     5701 agggccagaa gaactcacgc gagaggatga agaggatcga ggagggcatt aaggagctgg
     5761 ggtcccagat cctcaaggag cacccggtgg agaacacgca gctgcagaat gagaagctct
     5821 acctgtacta cctccagaat ggccgcgata tgtatgtgga ccaggagctg gatattaaca
     5881 ggctcagcga ttacgacgtc gatgctatcg ttccacagtc attcctgaag gatgactcca
     5941 ttgacaacaa ggtcctcacc aggtcggaca agaaccgggg caagtctgat aatgttcctt
     6001 cagaggaggt cgttaagaag atgaagaact actggcgcca gctcctgaat gccaagctga
     6061 tcacgcagcg gaagttcgat aacctcacaa aggctgagag gggcgggctc tctgagctgg
     6121 acaaggcggg cttcatcaag aggcagctgg tcgagacacg gcagatcact aagcacgttg
     6181 cgcagattct cgactcacgg atgaacacta agtacgatga gaatgacaag ctgatccgcg
     6241 aggtgaaggt catcaccctg aagtcaaagc tcgtctccga cttcaggaag gatttccagt
     6301 tctacaaggt tcgggagatc aacaattacc accatgccca tgacgcgtac ctgaacgcgg
     6361 tggtcggcac agctctgatc aagaagtacc caaagctcga gagcgagttc gtgtacgggg
     6421 actacaaggt ttacgatgtg aggaagatga tcgccaagtc ggagcaggag attggcaagg
     6481 ctaccgccaa gtacttcttc tactctaaca ttatgaattt cttcaagaca gagatcactc
     6541 tggccaatgg cgagatccgg aagcgccccc tcatcgagac gaacggcgag acgggggaga
     6601 tcgtgtggga caagggcagg gatttcgcga ccgtcaggaa ggttctctcc atgccacaag
     6661 tgaatatcgt caagaagaca gaggtccaga ctggcgggtt ctctaaggag tcaattaggc
     6721 ctaagcggaa cagcgacaag ctcatcgccc gcaagaagga ctgggatccg aagaagtacg
     6781 gcgggttcct gtggcccact gtggcctact cggtcctggt tgtggcgaag gttgagaagg
     6841 gcaagtccaa gaagctcaag agcgtgaagg agctgctggg gatcacgatt atggagcgct
     6901 ccagcttcga gaagaacccg atcgatttcc tggaggcgaa gggctacaag gaggtgaaga
     6961 aggacctgat cattaagctc cccaagtact cactcttcga gctggagaac ggcaggaagc
     7021 ggatgctggc ttccgctaag cagctgcaga aggggaacga gctggctctg ccgtccaagt
     7081 atgtgaactt cctctacctg gcctcccact acgagaagct caagggcagc cccgaggaca
     7141 acgagcagaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcattgagc
     7201 agatttccga gttctccaag cgcgtgatcc tggccgacgc gaatctggat aaggtcctct
     7261 ccgcgtacaa caagcaccgc gacaagccaa tcagggagca ggctgagaat atcattcatc
     7321 tcttcaccct gacgaggctc ggcgccccta gggctttcaa gtacttcgac acaactatcg
     7381 atccgaagca gtacaggagc actaaggagg tcctggacgc gaccctcatc caccagtcga
     7441 ttaccggcct ctacgagacg cgcatcgacc tgtctcagct cgggggcgac aagcggccag
     7501 cggcgacgaa gaaggcgggg caggcgaaga agaagaagtg agctcagagc tttcgttcgt
     7561 atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc gcacacacca
     7621 gaatcctact gagtttgagt attatggcat tgggaaaact gtttttcttg taccatttgt
     7681 tgtgcttgta atttactgtg ttttttattc ggttttcgct atcgaactgt gaaatggaaa
     7741 tggatggaga agagttaatg aatgatatgg tccttttgtt cattctcaaa ttaatattat
     7801 ttgttttttc tcttatttgt tgtgtgttga atttgaaatt ataagagata tgcaaacatt
     7861 ttgttttgag taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta atatgaggag
     7921 taaaacactt gtagttgtac cattatgctt attcactagg caacaaatat attttcagac
     7981 ctagaaaagc tgcaaatgtt actgaataca agtatgtcct cttgtgtttt agacatttat
     8041 gaactttcct ttatgtaatt ttccagaatc cttgtcagat tctaatcatt gctttataat
     8101 tatagttata ctcatggatt tgtagttgag tatgaaaata ttttttaatg cattttatga
     8161 cttgccaatt gttccggaac tctagataag cttaccggaa agggcgaatt cgcaactttg
     8221 tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     8281 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgccat
     8341 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     8401 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatc
//
