LOCUS       sequence-461810-        3281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For crRNA cloning with LbCas12a vectors for genome editing in 
            Arabidopsis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3281)
  AUTHORS   Xin C, Qiao D, Wang J, Sun W, Cao Z, Lu Y, Jiang Y, Chai Y, Wang XC,
            Chen QJ
  TITLE     Enhanced editing efficiency in Arabidopsis with a LbCas12a variant 
            harboring D156R and E795L mutations.
  JOURNAL   aBIOTECH. 2024 Mar 26;5(2):117-126. doi: 10.1007/s42994-024-00144-w.
            eCollection 2024 Jun.
  PUBMED    38978783
REFERENCE   2  (bases 1 to 3281)
  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"; doi: 
            "10.1007/s42994-024-00144-w"; journalName: "aBIOTECH"; date: 
            "2024-03-26- 26"; volume: "5"; issue: "2"; pages: "117-126"
COMMENT     Sequence Label: pAGC-Lb12-P1Bb
FEATURES             Location/Qualifiers
     source          1..3281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     ncRNA           complement(857..924)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1060..1076)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1060..1076)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1073..1095)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1084..1100
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1108..1114,1115..1132,1133..1138))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1153..1174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1291..1308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1462..2050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1542..1561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2221..3012,3013..3081))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2844..2863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3082..3186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3254..3272
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accagatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa
      421 tgcgtcgaga tcaccataca ttaccctgtt atccctattg gtctcacggc atgtcttcac
      481 accaccatga agacattcta cacttagtag aaattacaat ctcttagtcg actctaccaa
      541 tatataaaca gagcttctat tttcaactga acgatgtggg atgtttttga catcgtttac
      601 agcttaaacg ggctcgttta ttaaaagccc tcttctttcg atccatcaac attattggcc
      661 ttaagtaaaa caagcctctt tatgattgag aaaacgccgt cgttggtgca ccagagagaa
      721 gaaacaaaac agctagcgtt agataaactt taaaattgtt tgcatcctca tttgtctgtc
      781 aggctgcagt agtttggatt aaggatcatg agagctgaaa cacgcaaaaa agctgggaaa
      841 ttctatccaa aaaaaagtcc cattcgccat gccgaagcat gttgcccagc cggcgccagc
      901 gaggaggctg ggaccatgcc ggccaactgt cttcattggt tgtgaagaca tatcttgaga
      961 ccgtaactat aacggtccta aggtagcgaa gatattatta gattcggatg acgtgaacga
     1021 cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     1081 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     1141 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     1201 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1261 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1321 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1381 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1441 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1501 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1561 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1621 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1681 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1741 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1801 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1861 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1921 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1981 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2041 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2101 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2161 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2221 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2281 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2341 cagtgctgca atgataccgc gagagccacg ctcaccggct ccagatttat cagcaataaa
     2401 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2461 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2521 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2581 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2641 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2701 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2761 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2821 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2881 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2941 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3001 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3061 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3181 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     3241 attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
