LOCUS       sequence-119199-        4875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 Golden Gate Cassette: sgRNA cassette targeting PM19_3 in 
            barley.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4875)
  AUTHORS   Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N, 
            Uauy C, Harwood W
  TITLE     Induction of targeted, heritable mutations in barley and Brassica 
            oleracea using RNA-guided Cas9 nuclease.
  JOURNAL   Genome Biol. 2015 Nov 30;16(1):258. doi: 10.1186/s13059-015-0826-7.
  PUBMED    26616834
REFERENCE   2  (bases 1 to 4875)
  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: "Genome 
            Biol."; date: "2015-11-30"; volume: "16(1):258. doi"; pages: " 
            10.1186/s13059-015-0826-7"
COMMENT     Sequence Label: pICSL11058
FEATURES             Location/Qualifiers
     source          1..4875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        387..462
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(474..498)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    549..573
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(729..1877)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2213..2801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2293..2312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2972..3763,3764..3832))
                     /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     3595..3614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3833..3937)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4024..4655
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4747..4771
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggaggaccaa gcccgttatt ctgacagttc tggtgctcaa cacatttata tttatcaagg
       61 agcacattgt tactcactgc taggagggaa tcgaactagg aatattgatc agaggaacta
      121 cgagagagct gaagataact gccctctagc tctcactgat ctgggtcgca tagtgagatg
      181 cagcccacgt gagttcagca acggtctagc gctgggcttt taggcccgca tgatcgggct
      241 tttgtcgggt ggtcgacgtg ttcacgattg gggagagcaa cgcagcagtt cctcttagtt
      301 tagtcccacc tcgcctgtcc agcagagttc tgaccggttt ataaactcgc ttgctgcatc
      361 agacttggct ggcgttggtc gtaacagttt tagagctaga aatagcaagt taaaataagg
      421 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tagacccagc
      481 tttcttgtac aaagttggca ttacgctcag attgtcttct gcacgaagtg gtttaaacta
      541 tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat
      601 aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
      661 caaccacagg gttccccaga tcaggcgctg gctgctgaac ccccagccgg aactgacccc
      721 acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg
      781 ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg
      841 gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg
      901 tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc
      961 catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg
     1021 acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac
     1081 accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc
     1141 gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg
     1201 caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc
     1261 aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg
     1321 atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt
     1381 ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc
     1441 gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc
     1501 gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg
     1561 gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc
     1621 aaacctgccg cctcctgttc aagacgacgc gaacgctcca cggcggccga tggcgcgggc
     1681 agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc
     1741 atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg
     1801 gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg
     1861 tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg
     1921 tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg
     1981 gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc
     2041 ttgccctgca cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga
     2101 gagccaaaac acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg
     2161 cgccacatct aggatctgcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2221 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2281 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2341 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2401 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2461 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2521 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2581 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2641 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2701 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2761 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2821 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2881 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2941 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3001 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3061 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac
     3121 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3181 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3241 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3301 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3361 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3421 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3481 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3541 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3601 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3661 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3721 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3781 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3841 tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg
     3901 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     3961 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc
     4021 gttagcgggc cgggagggtt cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg
     4081 cgcacagggc gcagccctgg ttaaaaacaa ggtttataaa tattggttta aaagcaggtt
     4141 aaaagacagg ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc
     4201 tgcctgtgga cagcccctca aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc
     4261 cctcaagtgt caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat
     4321 accgcagggc acttatcccc aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc
     4381 aggcgttttc gccgatttgc gaggctggcc agctccacgt cgccggccga aatcgagcct
     4441 gcccctcatc tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc
     4501 ctcatctgtc agtgagggcc aagttttccg cgaggtatcc acaacgccgg cggccgcggt
     4561 gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca
     4621 gcccagcggc gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg atctgactga
     4681 tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct
     4741 ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac
     4801 attgcggacg tttttaatgt actggggtgg atgcagtggg ccccactctg tgaagacaat
     4861 tacgaattcc catgg
//
