LOCUS       sequence-481784-        3131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a gRNA expression cassette driven by the AtU6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3131)
  TITLE     Versatile molecular tools enabling customizable DNA methylation in 
            Arabidopsis thaliana
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3131)
  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            "Unpublished"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: pMD18T-AtU6-HV5
FEATURES             Location/Qualifiers
     source          1..3131
                     /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"
     misc_RNA        721..796
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(910..926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(910..926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(923..945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    934..950
                     /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(958..964,965..982,983..988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1003..1024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1141..1158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1312..1900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1392..1411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2071..2862,2863..2931))
                     /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     2694..2713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2932..3036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3104..3122
                     /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 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccgg tctccgatac attcggagtt
      421 tttgtatctt gtttcatagt ttgtcccagg attagaatga ttaggcatcg aaccttcaag
      481 aatttgattg aataaaacat cttcattctt aagatatgaa gataatcttc aaaaggcccc
      541 tgggaatctg aaagaagaga agcaggccca tttatatggg aaagaacaat agtatttctt
      601 atataggccc atttaagttg aaaacaatct tcaaaagtcc cacatcgctt agataagaaa
      661 acgaagctga gtttatatac agctagagtc gaagtagtga ttgggtcttc gagaagacct
      721 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      781 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
      841 tccgtagcgc gtgcgccaat tctgcagaca aattgatcga gacctaccga gctcgaattc
      901 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
      961 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1021 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1081 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1141 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1201 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1261 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1321 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1381 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1441 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     1501 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1561 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     1621 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1681 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1741 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1801 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1861 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1921 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     1981 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2041 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2101 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2161 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gggatccacg
     2221 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2281 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2341 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2401 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2461 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2521 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2581 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2641 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2701 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     2761 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2821 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2881 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2941 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3001 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3061 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     3121 gccctttcgt c
//
