LOCUS       sequence-413857-        5475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning protospacers for Cas9 editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5475)
  AUTHORS   Lawrenson T, Clarke M, Kirby R, Forner M, Steuernagel B, Harwood W
  TITLE     An Optimised CRISPR Cas9 and Cas12a Toolkit for Barley and Wheat
  JOURNAL   Research Square, 12 March 2024, PREPRINT
REFERENCE   2  (bases 1 to 5475)
  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: "Research 
            Square, 12 March 2024, PREPRINT"
COMMENT     Sequence Label: EC70199
FEATURES             Location/Qualifiers
     source          1..5475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    25..49
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(205..1353)
                     /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(1689..2277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1769..1788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2448..3239,3240..3308))
                     /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     3071..3090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3309..3413)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3500..4131
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4223..4247
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    4805..4826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4841..4846,4847..4864,4865..4871)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4879..4895
                     /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     4884..4906
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4903..4919
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4903..4919
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4915..5238
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    4932..4988
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4989..5006)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4989..5005)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4998..5020)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5214..5233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        5335..5410
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(5422..5446)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gaagtggttt aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa
       61 gagcgtttat tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc
      121 atttgtatgt gcatgccaac cacagggttc cccagatcag gcgctggctg ctgaaccccc
      181 agccggaact gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca
      241 ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg
      301 ccacttcttc acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag
      361 cgggtacggc tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag
      421 cttgcggtac ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat
      481 ttcctcgtcg atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa
      541 gcggtgcagc agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc
      601 cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga
      661 ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt
      721 gcgcttcgcg tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc
      781 gacgccggtg taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag
      841 cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt
      901 tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc
      961 cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt
     1021 tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat
     1081 ggtcatcgac ttcgccaaac ctgccgcctc ctgttcaaga cgacgcgaac gctccacggc
     1141 ggccgatggc gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc
     1201 cgtagcttgc tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac
     1261 ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg
     1321 cctgtatgcc ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac
     1381 tcacgccggg gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag
     1441 ataatccacc ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta
     1501 cttggtattc cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc
     1561 gtgggcctcg gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt
     1621 gtcgccggca tcgttgcgcc acatctagga tctgccagga accgtaaaaa ggccgcgttg
     1681 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1741 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1801 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1861 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1921 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1981 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2041 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2101 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2161 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2221 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2281 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2341 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2401 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2461 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2521 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2581 ataccgcgag aaccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2641 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2701 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2761 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2821 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2881 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2941 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3001 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3061 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3301 atactcatac tcttcctttt tcaatattat tgaagcattt atcaaggtta ttgtctcatg
     3361 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacgaat
     3421 tggccagcgc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg
     3481 gggatgggag gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc
     3541 ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt
     3601 ggtttaaaag caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg
     3661 caaatgctgg attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat
     3721 ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg
     3781 cccctcaagt gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg
     3841 aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc
     3901 ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa
     3961 gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa
     4021 cgccggcggc cgcggtgtct cgcacacggc ttcgacggcg tttctggcgc gtttgcaggg
     4081 ccatagacgg ccgccagccc agcggcgagg gcaaccagcc cggtgagcgt cgcaaaggag
     4141 atcctgatct gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt
     4201 cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta
     4261 gacaacttaa taacacattg cggacgtttt taatgtactg gggtggatgc agtgggcccc
     4321 actctgcttg aagacaacag aggcggcagg gagagtttta acattgacta gcgtgctgat
     4381 aatttgtgag aaataataat tgacaagtag atactgacat ttgagaagag cttctgaact
     4441 gttattagta acaaaaatgg aaagctgatg cacggaaaaa ggaaagaaaa agccatactt
     4501 ttttttaggt aggaaaagaa aaagccatac gagactgatg tctctcagat gggccgggat
     4561 ctgtctatct agcaggcagc agccctacca acctcacggg ccagcaatta cgagtccttc
     4621 taaaacgtcc cgccgagggc gcgtggccgt gctgtgcagc agcacgtcta acattagtcc
     4681 cacctcgcca gtttacaggg agcagaacca gcttataagc ggaggcgcgg caccaagaag
     4741 catgagaccg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4801 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4861 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     4921 atgattacgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc
     4981 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5041 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5101 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     5161 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     5221 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5281 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgacggt ctcagtttta
     5341 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     5401 gagtcggtgc tttttttcta gacccagctt tcttgtacaa agttggcatt acgctttact
     5461 gtgaagtctt cagtc
//
