LOCUS       sequence-436164-        8473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry plasmid (attL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8473)
  TITLE     Enhancing Cas12a and Cas9 mediated plant genome editing efficiency 
            and deletion size with the sbcB exonuclease
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8473)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pYPQ230-sbcB-N
FEATURES             Location/Qualifiers
     source          1..8473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             358..1149
                     /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      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1984..2001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2160..2187)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2279..2365)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2414..2436
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2428..2445
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2429..2445
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2461..2560
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4011..4058
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4119..4139
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4164..7844
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="SKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED
                     YKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEI
                     AKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSE
                     EAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE
                     FFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLS
                     DRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNG
                     PAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQL
                     QEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDL
                     LDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSK
                     DKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGN
                     YEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLI
                     DFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVE
                     EGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKE
                     ELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKIN
                     TEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDY
                     HSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGF
                     KNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQN
                     GFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDY
                     KNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQ
                     GDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNY
                     EAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSV
                     KH"
     CDS             7845..7892
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7923..8175
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    8220..8343
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8360..8379)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8361..8379)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8384..8400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8384..8400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
       61 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
      121 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      181 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
      241 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
      301 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacattatg
      361 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
      421 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      481 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      541 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      601 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      661 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      721 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      781 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      841 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      901 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      961 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     1021 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     1081 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     1141 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt agaaacgcaa
     2161 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     2221 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     2281 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     2341 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgctt
     2401 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2461 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
     2521 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgaattcgc ccttcaccat
     2581 ggcatatgat gaatgatgga aagcagcaat cgacgttcct tttccatgat tatgaaacgt
     2641 tcggaactca tcctgctctt gatagacctg ctcaatttgc agctattaga actgattctg
     2701 aatttaatgt tattggagaa cctgaagttt tttattgtaa gcctgctgat gattatcttc
     2761 cacaacctgg agctgttctt attactggaa ttactcctca agaagctaga gctaagggag
     2821 agaatgaagc agctttcgca gctagaattc attctctatt tactgttcct aagacttgta
     2881 ttcttggata taacaatgtt agattcgacg atgaagttac tagaaatatt ttttatagga
     2941 atttctatga tccttatgct tggtcttggc aacatgacaa ttctagatgg gatctacttg
     3001 atgttatgag agcttgttat gctcttagac ctgaaggaat taattggcct gaaaatgacg
     3061 atggacttcc ttctttcaga cttgaacatc ttactaaggc taatggaatt gaacattcta
     3121 atgctcatga tgcaatggct gatgtttatg caactattgc aatggctaag cttgttaaga
     3181 ctagacaacc tagacttttc gattatcttt ttactcatag aaataagcat aagcttatgg
     3241 ctcttattga tgttcctcaa atgaagcctc ttgttcatgt ttctggaatg ttcggagctt
     3301 ggagaggaaa tacttcttgg gttgcacctc ttgcttggca tcctgaaaat agaaacgctg
     3361 ttattatggt tgatcttgct ggagatattt ctcctctgct tgaactggat tctgatactc
     3421 ttagagaaag actttatact gctaagactg atcttggaga taatgccgct gttcctgtga
     3481 agcttgttca tattaataag tgtcctgttc ttgctcaagc gaatactctt agacctgaag
     3541 acgctgatag acttggaatt aatagacaac attgtcttga taatctgaag attcttagag
     3601 aaaatcctca agttagagaa aaggtcgttg ctattttcgc tgaagcggaa ccttttactc
     3661 cttctgataa tgttgatgct caactgtata atggattctt ttctgatgct gacagagctg
     3721 caatgaagat tgttcttgag actgaaccta gaaatctgcc tgctcttgat attacgttcg
     3781 ttgataagag aatcgaaaag ctgcttttca attatagagc taggaatttt cctggaactc
     3841 ttgattatgc tgaacaacaa agatggcttg aacataggag acaagttttc actcctgaat
     3901 ttcttcaagg atatgctgat gaacttcaaa tgcttgttca gcaatatgct gatgacaagg
     3961 aaaaggttgc tctccttaag gctctttggc aatatgctga agagattttt aaacgtcctg
     4021 ctgccaccaa aaaggccgga caggctaaga aaaagaagga gctctcagga tctgagaccc
     4081 caggcacaag tgaatcagct acacctgagt ccatggctcc taagaagaag cggaaggttg
     4141 gtattcacgg ggtgcctgcg gcttcaaagc tcgagaaatt caccaactgt tattcgttga
     4201 gcaaaacact gcggtttaaa gcgattccag tcggcaagac tcaagagaat atagacaata
     4261 agcggctgtt ggtggaagat gaaaagcgcg cggaagacta caaaggggtg aagaagttgt
     4321 tggacagata ctacctctct tttatcaatg atgtcttgca ctcaatcaaa ttgaagaatc
     4381 tgaacaacta catctccctc ttcagaaaga aaacaaggac agaaaaggag aataaggaac
     4441 ttgaaaattt ggagatcaat ctgaggaaag agatcgcgaa agcctttaaa ggcaacgaag
     4501 gatacaaaag tctgttcaag aaggatataa ttgagacaat tttgccagag ttcctcgatg
     4561 acaaggacga gattgcgctg gtcaattcgt tcaacggatt cacaacagca ttcacaggct
     4621 tctttgataa tcgggaaaat atgttctctg aggaggcaaa gtccacttct attgcgttca
     4681 ggtgtatcaa tgagaatctc actaggtaca tttccaacat ggatatcttt gagaaggttg
     4741 acgcaatttt tgacaagcac gaagttcagg agattaagga gaagatcctc aattccgatt
     4801 atgacgttga ggacttcttc gaaggtgagt tttttaattt cgtgctcact caagagggta
     4861 tcgacgtgta taatgcgatc atcggtgggt tcgtgactga gtccggtgaa aagattaagg
     4921 gattgaacga gtatatcaac ctttacaacc aaaagacgaa acagaagctg ccaaagttca
     4981 agcctcttta caaacaggtt ctttcagacc gcgagtcact ctcgttctat ggggagggct
     5041 acacttcgga tgaggaagtc ctggaggtgt tcaggaatac tctcaataag aattcggaga
     5101 ttttctcttc tataaaaaaa ctggaaaagt tgtttaagaa ttttgacgaa tactctagcg
     5161 ccggcatatt tgtgaaaaac ggcccggcca tatcaacgat aagtaaagat atcttcggcg
     5221 aatggaacgt gatcagagac aaatggaacg cggagtatga cgatattcac ctgaagaaga
     5281 aggctgtcgt aacggagaag tacgaggatg atcgcaggaa aagcttcaaa aagatcggaa
     5341 gtttcagcct ggaacagttg caggagtatg ctgacgccga tcttagcgtc gtcgagaagt
     5401 tgaaggagat aatcatccaa aaggtcgacg agatatataa agtctatgga tcaagtgaaa
     5461 aactgttcga cgccgacttc gttttggaga agtccctgaa gaagaacgac gctgttgttg
     5521 ccattatgaa ggatctgctc gacagcgtga agagtttcga gaactatatt aaggcttttt
     5581 tcggggaggg gaaggagact aacagagatg agtccttcta cggagacttc gtcctcgcgt
     5641 acgatatact ccttaaggta gaccacatct acgacgcaat cagaaattac gtgacacaaa
     5701 agccgtacag caaggacaag ttcaaactct acttccagaa cccccagttc atgggcggct
     5761 gggacaagga caaggaaacg gattacaggg ctacgatcct gaggtatggt tcaaaatact
     5821 acttggcgat tatggacaag aagtacgcca agtgtctcca gaagattgac aaagacgatg
     5881 tcaatggcaa ttatgagaag atcaactaca agctgcttcc gggtccgaac aagatgctcc
     5941 caaaggtttt cttcagcaag aaatggatgg cctactataa cccaagcgag gacatccaga
     6001 agatttataa gaacggtacg ttcaagaagg gcgacatgtt caatcttaac gactgtcaca
     6061 agctgatcga cttcttcaaa gactcaatta gccggtaccc aaagtggtct aacgcctatg
     6121 acttcaactt ttcggaaacc gagaagtaca aggatatagc cggattttat agagaggtgg
     6181 aagagcaggg ctacaaggtg tcattcgagt ccgccagcaa gaaggaagtg gacaagctcg
     6241 tggaagaggg taagctctac atgttccaga tttataataa agactttagc gataagagcc
     6301 acgggacacc taatctccac acaatgtatt tcaagctgct cttcgacgag aataaccacg
     6361 gccaaatcag gttgtcagga ggggctgaac tcttcatgcg gcgcgctagc cttaagaagg
     6421 aggagcttgt agtccaccct gcgaatagtc caattgcgaa taagaacccg gacaatccta
     6481 aaaagactac aacattgagc tacgacgtgt acaaggataa gaggttttcc gaggatcagt
     6541 acgagctcca catcccgatt gcgatcaaca agtgcccaaa gaatattttc aagataaaca
     6601 cagaggtgcg tgtactcctg aagcatgacg acaatcctta cgtcattggg attgatcggg
     6661 gcgagaggaa cctcctctat attgtggtgg tggacgggaa ggggaacata gtcgaacagt
     6721 actcccttaa cgaaataatt aacaatttca acggcatccg tatcaagacc gactaccatt
     6781 cgttgctgga caagaaggag aaggagagat ttgaggcgcg gcaaaattgg acaagtatcg
     6841 agaacatcaa ggaactcaaa gcaggttata tctctcaagt tgtgcataag atatgcgagc
     6901 tggttgagaa gtatgacgca gtgatcgctc ttgaggacct caactcgggc tttaagaatt
     6961 ctagagttaa agtggagaag caggtctatc aaaagttcga gaagatgctt atagataagc
     7021 tcaactacat ggtcgataag aaatcgaacc catgtgccac cggcggcgca ctcaaaggtt
     7081 accaaataac aaacaaattc gagtccttca aatcgatgag tactcagaat gggttcatat
     7141 tttatatacc ggcgtggctt acgtctaaga tcgacccgtc aactggtttt gtcaacctgt
     7201 tgaagacgaa atacacgtcc attgccgatt cgaaaaagtt catatctagt tttgatcgta
     7261 ttatgtacgt cccagaggaa gatcttttcg agtttgctct cgactacaaa aacttttcgc
     7321 ggaccgatgc ggattacatt aaaaaatgga aactctattc gtacggcaac agaatcagga
     7381 tttttcgcaa ccctaagaag aataacgtct ttgattggga ggaagtttgc ttgactagcg
     7441 cgtacaagga gctctttaat aagtatggca ttaactacca acagggtgat atcagagcac
     7501 tgctttgcga acaatctgac aaggctttct actcatcctt catggctttg atgagcctga
     7561 tgctccagat gagaaattca attacaggca gaaccgacgt ggatttcttg atctccccgg
     7621 ttaaaaattc tgatggcatc ttttacgata gcaggaacta tgaagcgcaa gagaatgcga
     7681 ttctgccaaa aaatgcagac gccaacggtg cctataacat cgccaggaaa gtcctgtggg
     7741 cgatcggcca gttcaaaaag gccgaagacg aaaaattgga caaggtcaaa atcgctatca
     7801 gcaacaaaga gtggctggag tatgctcaga catccgtaaa gcataagcgt cctgctgcca
     7861 ccaaaaaggc cggacaggct aagaaaaaga agtgagacga ctagtggcgg ccgccgacgt
     7921 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg
     7981 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat
     8041 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat
     8101 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat
     8161 ctatgttact agatcgggaa ttgatccccc ctcgacagct tccggaaagg gcgaattcgc
     8221 aactttgtat acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa
     8281 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact
     8341 atgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     8401 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     8461 atcatgcctc ctc
//
