LOCUS       sequence-413831-        3445 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 3445)
  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 3445)
  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: EC67894
FEATURES             Location/Qualifiers
     source          1..3445
                     /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    900..921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(936..941,942..959,960..966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    974..990
                     /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     979..1001
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     998..1014
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1010..1333
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1027..1083
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1084..1100)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1309..1328)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1588..1605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1698..2286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1778..1797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2457..3248,3249..3317))
                     /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     3080..3099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3318..3422)
                     /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 aatgccgacc aagcccgtta ttctgacagt tctggtgctc
      421 aacacattta tatttatcaa ggagcacatt gttactcact gctaggaggg aatcgaacta
      481 ggaatattga tcagaggaac tacgagagag ctgaagataa ctgccctcta gctctcactg
      541 atctgggtcg catagtgaga tgcagcccac gtgagttcag caacggtcta gcgctgggct
      601 tttaggcccg catgatcggg cttttgtcgg gtggtcgacg tgttcacgat tggggagagc
      661 aacgcagcag ttcctcttag tttagtccca cctcgcctgt ccagcagagt tctgaccggt
      721 ttataaactc gcttgctgca tcagacttgc cagtggtcta gtggtagaat agtaccctgc
      781 cacggtacag acccgggttc gattcccggc tggaaataat ttctactaag tgtagattga
      841 gaccgcagct ggcacgacag gtttgccgac tggaaagcgg gcagtgagcg caacgcaatt
      901 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      961 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1021 tacgccaagc ttgcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa
     1081 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1141 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1201 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     1261 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     1321 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     1381 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg acggtctcat aatttctact
     1441 aagtgtagat ttttttgcaa ttgtcttcct ggcctgggcc tcatgggcct tccgctcact
     1501 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taacatggtc atagctgttt
     1561 ccttgcgtat tgggcgctct ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1621 ggtaaagcct ggggtgccta atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1681 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1741 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1801 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1861 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1921 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1981 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2041 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2101 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2161 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2221 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2281 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2341 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2401 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2461 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2521 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2581 gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag
     2641 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2701 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2761 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2821 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2881 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2941 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3001 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3061 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3121 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3181 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3241 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3301 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3361 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3421 cgcacatttc cccgaaaagt gccac
//
