LOCUS       sequence-413858-        3505 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 3505)
  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 3505)
  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: EC70122
FEATURES             Location/Qualifiers
     source          1..3505
                     /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           794..830
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    914..935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(950..955,956..973,974..980)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    988..1004
                     /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     993..1015
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1012..1028
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1024..1347
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1041..1097
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1098..1114)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1323..1342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1444..1511
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1648..1665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1758..2346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1838..1857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2517..3308,3309..3377))
                     /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     3140..3159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3378..3482)
                     /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 aggccgcatc gcatgaagac aattacggcg gcagggagag ttttaacatt gactagcgtg
      421 ctgataattt gtgagaaata ataattgaca agtagatact gacatttgag aagagcttct
      481 gaactgttat tagtaacaaa aatggaaagc tgatgcacgg aaaaaggaaa gaaaaagcca
      541 tacttttttt taggtaggaa aagaaaaagc catacgagac tgatgtctct cagatgggcc
      601 gggatctgtc tatctagcag gcagcagccc taccaacctc acgggccagc aattacgagt
      661 ccttctaaaa cgtcccgccg agggcgcgtg gccgtgctgt gcagcagcac gtctaacatt
      721 agtcccacct cgccagttta cagggagcag aaccagctta taagcggagg cgcggcacca
      781 agaagcaaaa ttactgatga gtccgtgagg acgaaacgag taagctcgtc taatttctac
      841 taagtgtaga ttgagaccgc agctggcacg acaggtttgc cgactggaaa gcgggcagtg
      901 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      961 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     1021 gctatgacca tgattacgcc aagcttgcat gcctgcaggt cgactctaga ggatccccgg
     1081 gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     1141 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     1201 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     1261 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     1321 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     1381 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgacggtc
     1441 tcaggccggc atggtcccag cctcctcgct ggcgccggct gggcaacatg cttcggcatg
     1501 gcgaatggga ctttttcaga ttgtcttcct ggcctgggcc tcatgggcct tccgctcact
     1561 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taacatggtc atagctgttt
     1621 ccttgcgtat tgggcgctct ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1681 ggtaaagcct ggggtgccta atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1741 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1801 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1861 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1921 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1981 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2041 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2101 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2161 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2221 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2281 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2341 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2401 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2461 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2521 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2581 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2641 gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag
     2701 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2761 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2821 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2881 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2941 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3001 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3061 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3121 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3181 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3241 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3301 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3361 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3421 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3481 cgcacatttc cccgaaaagt gccac
//
