LOCUS       sequence-331270-        3463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GoldenGate (MoClo) Level 1 Position 3 TaU6 guide acceptor plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3463)
  AUTHORS   Smedley MA, Hayta S, Clarke M, Harwood WA
  TITLE     CRISPR-Cas9 Based Genome Editing in Wheat.
  JOURNAL   Curr Protoc. 2021 Mar;1(3):e65. doi: 10.1002/cpz1.65.
  PUBMED    33687760
REFERENCE   2  (bases 1 to 3463)
  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: "Curr 
            Protoc."; date: "2021-03"; volume: "1(3):e65. doi"; pages: " 
            10.1002/cpz1.65"
COMMENT     Sequence Label: Level 1 P3 TaU6 guide acceptor
FEATURES             Location/Qualifiers
     source          1..3463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     28..47
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        28..46
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    524..545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(560..565,566..583,584..590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    598..614
                     /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)."
     primer_bind     603..625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     622..638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             634..957
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    651..707
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(708..724)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(933..952)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        1054..1129
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1141..1165)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1314..1331)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1424..2012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1504..1523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2183..2974,2975..3043))
                     /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     2806..2825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3044..3148)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgacgtaa tacgactcac tatagggcga attggcggaa
       61 ggccgtcaag gccgcatcac tctgtggaag acaaactaga ccaagcccgt tattctgaca
      121 gttctggtgc tcaacacatt tatatttatc aaggagcaca ttgttactca ctgctaggag
      181 ggaatcgaac taggaatatt gatcagagga actacgagag agctgaagat aactgccctc
      241 tagctctcac tgatctgggt cgcatagtga gatgcagccc acgtgagttc agcaacggtc
      301 tagcgctggg cttttaggcc cgcatgatcg ggcttttgtc gggtggtcga cgtgttcacg
      361 attggggaga gcaacgcagc agttcctctt agtttagtcc cacctcgcct gtccagcaga
      421 gttctgaccg gtttataaac tcgcttgctg catcagactt gtgagaccgc agctggcacg
      481 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      541 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      601 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat
      661 gcctgcaggt cgactctaga ggatccccgg gtaccgagct cgaattcact ggccgtcgtt
      721 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      781 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      841 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
      901 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
      961 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     1021 gcatccgctt acagacaagc tgtgacggtc tcagttttag agctagaaat agcaagttaa
     1081 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctag
     1141 acccagcttt cttgtacaaa gttggcatta cgctttactt acaagtcttc cacgaagtgc
     1201 tgggcctcat gggccttccg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1261 ctgcattaac atggtcatag ctgtttcctt gcgtattggg cgctctccgc ttcctcgctc
     1321 actgactcgc tgcgctcggt cgttcgggta aagcctgggg tgcctaatga gcaaaaggcc
     1381 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1441 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1501 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1561 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1621 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1681 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1741 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1801 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1861 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1921 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1981 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2041 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2101 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2161 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2221 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2281 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaacca cgctcaccgg
     2341 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2401 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2461 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2521 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2581 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2641 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2701 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2761 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2821 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2881 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2941 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3001 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3061 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3121 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     3181 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3241 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3301 gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     3361 gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     3421 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gtt
//
