LOCUS       sequence-266587-        3181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector for 2nd crRNA cloning with LbCas12a crRNA 
            scaffold flanked by HH and HDV ribozymes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3181)
  AUTHORS   Zhang Y, Ren Q, Tang X, Liu S, Malzahn AA, Zhou J, Wang J, Yin D, 
            Pan C, Yuan M, Huang L, Yang H, Zhao Y, Fang Q, Zheng X, Tian L, 
            Cheng Y, Le Y, McCoy B, Franklin L, Selengut JD, Mount SM, Que Q, 
            Zhang Y, Qi Y
  TITLE     Expanding the scope of plant genome engineering with Cas12a 
            orthologs and highly multiplexable editing systems.
  JOURNAL   Nat Commun. 2021 Mar 29;12(1):1944. doi: 10.1038/s41467-021-22330-w.
  PUBMED    33782402
REFERENCE   2  (bases 1 to 3181)
  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: "Nat 
            Commun."; date: "2021-03-29"; volume: "12(1):1944. doi"; pages: " 
            10.1038/s41467-021-22330-w"
COMMENT     Sequence Label: pYPQ132-STU-Lb
FEATURES             Location/Qualifiers
     source          1..3181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(196..201,202..218,219..224)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             272..1462
                     /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(624..643)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     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"
     ncRNA           1771..1807
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     ncRNA           1861..1928
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     rep_origin      2030..2618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2519..2538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2948..2975)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3067..3153)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
       61 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      121 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      181 aattaattct catgtttgac agcttatcat cgattagctt taatgcggta gtttatcaca
      241 gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat
      301 cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg
      361 cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg tgctgctagc
      421 gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt ccgaccgctt
      481 tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact acgcgatcat
      541 ggcgaccaca cccgtcctgt ggattctcta cgccggacgc atcgtggccg gcatcaccgg
      601 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
      661 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
      721 cgggggactg ttgggcgcca tctccttaca tgcaccattc cttgcggcgg cggtgctcaa
      781 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgccg
      841 acccatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
      901 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
      961 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     1021 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     1081 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg
     1141 ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct
     1201 cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga
     1261 cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat
     1321 tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc
     1381 atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     1441 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     1501 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttga
     1561 tcggggaaga acagtatgtc gagctatttt ttgacttact ggggatcaag cctgattggg
     1621 agaaaataaa atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     1681 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     1741 tgcctcctct agaggtctcc catgaaatta ctgatgagtc cgtgaggacg aaacgagtaa
     1801 gctcgtctaa tttctactaa gtgtagatga gacggagctc agtctgaccg cggcgtctct
     1861 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacatgctt cggcatggcg
     1921 aatgggacgg accgagaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg
     1981 cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2041 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2101 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2161 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2221 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2281 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2341 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2401 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2461 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2521 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2581 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2641 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2701 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2761 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2821 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2881 aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag
     2941 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     3001 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     3061 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     3121 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
     3181 t
//
