LOCUS       sequence-222710-        3463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA in barley.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3463)
  AUTHORS   Lawrenson T, Harwood WA
  TITLE     Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1900:217-232. doi: 
            10.1007/978-1-4939-8944-7_14.
  PUBMED    30460568
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: "Methods Mol
            Biol."; date: "2019"; volume: "1900:217-232. doi"; pages: " 
            10.1007/978-1-4939-8944-7_14"
COMMENT     Sequence Label: pHvL1P3GA
FEATURES             Location/Qualifiers
     source          1..3463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..36
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     47..66
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        47..65
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    543..564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(579..584,585..602,603..609)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    617..633
                     /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     622..644
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     641..657
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             653..976
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    670..726
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(727..743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(952..971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        1073..1148
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1160..1184)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1333..1350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1443..2031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1523..1542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2202..2993,2994..3062))
                     /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     2825..2844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3063..3167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgacgtaat acgactcact
       61 atagggcgaa ttggcggaag gccgtcaagg ccgcatcact ctgtggaaga caaactagac
      121 caagcccgtt attctgacag ttctggtgct caacacattt atatttatca aggagcacat
      181 tgttactcac tgctaggagg gaatcgaact aggaatattg atcagaggaa ctacgagaga
      241 gctgaagata actgccctct agctctcact gatctgggtc gcatagtgag atgcagccca
      301 cgtgagttca gcaacggtct agcgctgggc ttttaggccc gcatgatcgg gcttttgtcg
      361 ggtggtcgac gtgttcacga ttggggagag caacgcagca gttcctctta gtttagtccc
      421 acctcgcctg tccagcagag ttctgaccgg tttataaact cgcttgctgc atcagacttg
      481 tgagaccgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      541 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      601 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      661 gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc
      721 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
      781 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
      841 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
      901 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
      961 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     1021 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacggtct cagttttaga
     1081 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     1141 gtcggtgctt tttttctaga cccagctttc ttgtacaaag ttggcattac gctttactta
     1201 caagtcttcc acgaagtgct gggcctcatg ggccttccgc tcactgcccg ctttccagtc
     1261 gggaaacctg tcgtgccagc tgcattaaca tggtcatagc tgtttccttg cgtattgggc
     1321 gctctccgct tcctcgctca ctgactcgct gcgctcggtc gttcgggtaa agcctggggt
     1381 gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1441 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1501 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1561 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1621 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1681 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1741 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1801 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1861 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1921 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1981 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2041 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2101 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2161 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2221 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2281 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2341 cgagaaccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2401 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2461 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2521 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2581 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2641 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2701 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2761 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2821 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2881 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2941 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3001 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3061 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3121 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3181 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3241 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3301 aatagaccga gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg
     3361 cgcaactgtt gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3421 agggggatgt gctgcaaggc gattaagttg ggtaacgcca ggg
//
