LOCUS       sequence-272943-        3503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector; empty vector to clone the 1st LbCas12a 
            crRNA under OsU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3503)
  AUTHORS   Li S, Zhang Y, Xia L, Qi Y
  TITLE     CRISPR-Cas12a enables efficient biallelic gene targeting in rice.
  JOURNAL   Plant Biotechnol J. 2019 Nov 15. doi: 10.1111/pbi.13295.
  PUBMED    31730252
REFERENCE   2  (bases 1 to 3503)
  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: "Plant 
            Biotechnol J. 2019 Nov 15. doi: 10.1111/pbi.13295."
COMMENT     Sequence Label: pYPQ131C-RZ-Lb
FEATURES             Location/Qualifiers
     source          1..3503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(107..134)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(226..312)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(536..541,542..558,559..564)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             612..1802
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(964..983)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1976..1995)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1977..1995)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2000..2016)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2000..2016)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     ncRNA           2411..2447
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     ncRNA           2501..2568
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     rep_origin      2692..3280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3181..3200
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
       61 caattaatac gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt
      121 caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca
      181 ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag
      241 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt
      301 tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt ctgcattaat gaatcggcca
      361 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      421 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      481 gttatccaca gaatcagggg ataacgcagg aaagaacatg aattaattct catgtttgac
      541 agcttatcat cgattagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc
      601 aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg
      661 atgctgtagg cataggcttg gttatgccgg tactgccggg cctcttgcgg gatatcgtcc
      721 attccgacag catcgccagt cactatggcg tgctgctagc gctatatgcg ttgatgcaat
      781 ttctatgcgc acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc
      841 tcgcttcgct acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt
      901 ggattctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
      961 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     1021 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     1081 tctccttaca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     1141 gctgcttcct aatgcaggag tcgcataagg gagagcgccg acccatgccc ttgagagcct
     1201 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1261 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     1321 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     1381 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     1441 agcaggccat tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg
     1501 cgacgcgagg ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga
     1561 tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc
     1621 aaggatcgct cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg
     1681 cgatttatgc cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc
     1741 tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct
     1801 gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca
     1861 attcttgcgg agaactgtga atgcgcaaac caacccttga tcggggaaga acagtatgtc
     1921 gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa atatccccta
     1981 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     2041 tctctgatgt tacattgcac aagataaaaa tatatcatca tgcctcctct agaggtctcg
     2101 ctatggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttgcccttgg
     2161 atcatgaacc aacggcctgg ctgtatttgg tggttgtgta gggagatggg gagaagaaaa
     2221 gcccgattct cttcgctgtg atgggctgga tgcatgcggg ggagcgggag gcccaagtac
     2281 gtgcacggtg agcggcccac agggcgagtg tgagcgcgag aggcgggagg aacagtttag
     2341 taccacattg cccagctaac tcgaacgcga ccaacttata aacccgcgcg ctgtcgcttg
     2401 tgtgaaatta ctgatgagtc cgtgaggacg aaacgagtaa gctcgtctaa tttctactaa
     2461 gtgtagatga gacggagctc agtctgaccg cggcgtctct ggccggcatg gtcccagcct
     2521 cctcgctggc gccggctggg caacatgctt cggcatggcg aatgggacca tgggtggctc
     2581 gagggtctcc catgggagac cctcgagcca cccatgacca aaatccctta acgtgagtta
     2641 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2821 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     3481 gcctctcccc gcgcgttggc cga
//
