LOCUS       sequence-413828-        3435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning protospacers for Cas12a editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3435)
  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 3435)
  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: EC67895
FEATURES             Location/Qualifiers
     source          1..3435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    895..916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(931..936,937..954,955..961)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    969..985
                     /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     974..996
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     993..1009
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1005..1328
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1022..1078
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1079..1095)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1304..1323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1578..1595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1688..2276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1768..1787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2447..3238,3239..3307))
                     /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     3070..3089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3308..3412)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccgcatg aagacaagca agaccaagcc cgttattctg acagttctgg tgctcaacac
      421 atttatattt atcaaggagc acattgttac tcactgctag gagggaatcg aactaggaat
      481 attgatcaga ggaactacga gagagctgaa gataactgcc ctctagctct cactgatctg
      541 ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg gtctagcgct gggcttttag
      601 gcccgcatga tcgggctttt gtcgggtggt cgacgtgttc acgattgggg agagcaacgc
      661 agcagttcct cttagtttag tcccacctcg cctgtccagc agagttctga ccggtttata
      721 aactcgcttg ctgcatcaga cttgccagtg gtctagtggt agaatagtac cctgccacgg
      781 tacagacccg ggttcgattc ccggctggaa ataatttcta ctaagtgtag attgagaccg
      841 cagctggcac gacaggtttg ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      901 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      961 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     1021 caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc tcgaattcac
     1081 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     1141 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     1201 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     1261 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     1321 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     1381 gtctgctccc ggcatccgct tacagacaag ctgtgacggt ctcataattt ctactaagtg
     1441 tagatttttt tactattgtc ttcctgggcc tcatgggcct tccgctcact gcccgctttc
     1501 cagtcgggaa acctgtcgtg ccagctgcat taacatggtc atagctgttt ccttgcgtat
     1561 tgggcgctct ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg ggtaaagcct
     1621 ggggtgccta atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1681 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1741 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1801 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1861 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1921 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1981 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2041 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2101 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2161 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2221 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2281 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2341 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2401 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2461 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2521 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2581 taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2641 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2701 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2761 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2821 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2881 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2941 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3001 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3061 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3121 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3181 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3241 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3301 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3361 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3421 cccgaaaagt gccac
//
