LOCUS       sequence-222718-        3946 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 3946)
  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 3946)
  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: pHvL1P4GA
FEATURES             Location/Qualifiers
     source          1..3946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     19..36
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     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    584..605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(620..625,626..643,644..650)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    658..674
                     /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)."
     RBS             692..703
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             710..1387
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1432..1503
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1519..1546
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(1643..1667)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1816..1833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1926..2514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2006..2025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2685..3476,3477..3545))
                     /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     3308..3327
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3546..3650)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgacgtaat acgactcact
       61 atagggcgaa ttggcggaag gccgtcaagg ccgcatcact ctgtggaaga caattacgac
      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 atcagacgag
      481 acgcaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      541 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      601 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      661 tgagcggata acaatttcac acatactaga gaaagaggag aaatactaga tggcttcctc
      721 cgaggacgtt atcaaagagt tcatgcgttt caaagttcgt atggaaggtt ccgttaacgg
      781 tcacgagttc gaaatcgaag gtgaaggtga aggtcgtccg tacgaaggta cccagaccgc
      841 taaactgaaa gttaccaaag gtggtccgct gccgttcgct tgggacatcc tgtccccgca
      901 gttccagtac ggttccaaag cttacgttaa acacccggct gacatcccgg actacctgaa
      961 actgtccttc ccggaaggtt tcaaatggga acgtgttatg aacttcgagg acggtggtgt
     1021 tgttaccgtt acccaggact cctccctgca agacggtgag ttcatctaca aagttaaact
     1081 gcgtggtacc aacttcccgt ccgacggtcc ggttatgcag aaaaaaacca tgggttggga
     1141 agcttccacc gaacgtatgt acccggagga cggtgctctg aaaggtgaaa tcaaaatgcg
     1201 tctgaaactg aaagacggtg gtcactacga cgctgaagtt aaaaccacct acatggctaa
     1261 aaaaccggtt cagctgccgg gtgcttacaa aaccgacatc aaactggaca tcacctccca
     1321 caacgaggac tacaccatcg ttgaacagta cgaacgtgct gaaggtcgtc actccaccgg
     1381 tgcttaataa cgctgatagt gctagtgtag atcgctacta gagccaggca tcaaataaaa
     1441 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     1501 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacgtctctt
     1561 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1621 cgagtcggtg ctttttttct agacccagct ttcttgtaca aagttggcat tacgctttac
     1681 gggaaagtct tccacgaagt gctgggcctc atgggccttc cgctcactgc ccgctttcca
     1741 gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat agctgtttcc ttgcgtattg
     1801 ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggg taaagcctgg
     1861 ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1921 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1981 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2041 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2101 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2161 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2221 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2281 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2341 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2401 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2461 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2521 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2581 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2641 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2701 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2761 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2821 ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2881 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2941 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3001 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3061 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3121 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3181 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3241 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3301 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3361 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3421 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3481 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3541 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3601 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3661 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     3721 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3781 aagaatagac cgagataggg ttgagtggcc gctacagggc gctcccattc gccattcagg
     3841 ctgcgcaact gttgggaagg gcgtttcggt gcgggcctct tcgctattac gccagctggc
     3901 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccaggg
//
