LOCUS       sequence-413847-        3495 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 3495)
  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 3495)
  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: EC70121
FEATURES             Location/Qualifiers
     source          1..3495
                     /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"
     ncRNA           789..825
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    909..930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(945..950,951..968,969..975)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    983..999
                     /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     988..1010
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1007..1023
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1019..1342
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1036..1092
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1093..1109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1318..1337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1439..1506
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1638..1655)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1748..2336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1828..1847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2507..3298,3299..3367))
                     /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     3130..3149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3368..3472)
                     /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 aagacaaact aggcggcagg gagagtttta acattgacta gcgtgctgat
      421 aatttgtgag aaataataat tgacaagtag atactgacat ttgagaagag cttctgaact
      481 gttattagta acaaaaatgg aaagctgatg cacggaaaaa ggaaagaaaa agccatactt
      541 ttttttaggt aggaaaagaa aaagccatac gagactgatg tctctcagat gggccgggat
      601 ctgtctatct agcaggcagc agccctacca acctcacggg ccagcaatta cgagtccttc
      661 taaaacgtcc cgccgagggc gcgtggccgt gctgtgcagc agcacgtcta acattagtcc
      721 cacctcgcca gtttacaggg agcagaacca gcttataagc ggaggcgcgg caccaagaag
      781 caaaattact gatgagtccg tgaggacgaa acgagtaagc tcgtctaatt tctactaagt
      841 gtagattgag accgcagctg gcacgacagg tttgccgact ggaaagcggg cagtgagcgc
      901 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
      961 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     1021 gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc cccgggtacc
     1081 gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     1141 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     1201 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     1261 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     1321 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     1381 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga cggtctcagg
     1441 ccggcatggt cccagcctcc tcgctggcgc cggctgggca acatgcttcg gcatggcgaa
     1501 tgggactttt tttacttgtc ttcctgggcc tcatgggcct tccgctcact gcccgctttc
     1561 cagtcgggaa acctgtcgtg ccagctgcat taacatggtc atagctgttt ccttgcgtat
     1621 tgggcgctct ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg ggtaaagcct
     1681 ggggtgccta atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1741 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1801 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1861 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1921 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1981 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2041 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2101 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2161 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2221 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2281 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2641 taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3481 cccgaaaagt gccac
//
