LOCUS       sequence-266585-        3180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector for 2nd crRNA cloning with AsCas12a crRNA 
            scaffold flanked by HH and HDV ribozymes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3180)
  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 3180)
  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-As
FEATURES             Location/Qualifiers
     source          1..3180
                     /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           1860..1927
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     rep_origin      2029..2617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2518..2537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2947..2974)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3066..3152)
                     /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 tttctactct tgtagatgag acggagctca gtctgaccgc ggcgtctctg
     1861 gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacatgcttc ggcatggcga
     1921 atgggacgga ccgagaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc
     1981 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2041 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2101 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2161 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2221 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2281 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2341 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2401 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2461 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2521 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2581 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2641 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2701 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2761 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     2821 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2881 agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga
     2941 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
     3001 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
     3061 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     3121 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
//
