LOCUS       sequence-413833-        3451 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 3451)
  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 3451)
  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: EC70136
FEATURES             Location/Qualifiers
     source          1..3451
                     /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    906..927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(942..947,948..965,966..972)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    980..996
                     /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     985..1007
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1004..1020
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1016..1339
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1033..1089
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1090..1106)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1315..1334)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1594..1611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1704..2292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1784..1803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2463..3254,3255..3323))
                     /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     3086..3105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3324..3428)
                     /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 gcattctgtg aagacaatgt gtgaccaagc ccgttattct gacagttctg
      421 gtgctcaaca catttatatt tatcaaggag cacattgtta ctcactgcta ggagggaatc
      481 gaactaggaa tattgatcag aggaactacg agagagctga agataactgc cctctagctc
      541 tcactgatct gggtcgcata gtgagatgca gcccacgtga gttcagcaac ggtctagcgc
      601 tgggctttta ggcccgcatg atcgggcttt tgtcgggtgg tcgacgtgtt cacgattggg
      661 gagagcaacg cagcagttcc tcttagttta gtcccacctc gcctgtccag cagagttctg
      721 accggtttat aaactcgctt gctgcatcag acttgccagt ggtctagtgg tagaatagta
      781 ccctgccacg gtacagaccc gggttcgatt cccggctgga aataatttct actaagtgta
      841 gattgagacc gcagctggca cgacaggttt gccgactgga aagcgggcag tgagcgcaac
      901 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
      961 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     1021 catgattacg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc gggtaccgag
     1081 ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1141 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1201 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     1261 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     1321 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     1381 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacgg tctcataatt
     1441 tctactaagt gtagattttt ttgagcttgt cttcctggcc tgggcctcat gggccttccg
     1501 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaac atggtcatag
     1561 ctgtttcctt gcgtattggg cgctctccgc ttcctcgctc actgactcgc tgcgctcggt
     1621 cgttcgggta aagcctgggg tgcctaatga gcaaaaggcc agcaaaaggc caggaaccgt
     1681 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1741 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1801 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1861 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1921 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1981 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2041 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2101 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2161 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2221 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2281 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2341 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2401 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2461 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2521 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2581 cccagtgctg caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata
     2641 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2701 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2761 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2821 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2881 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2941 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3001 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3061 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3121 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3181 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3241 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3301 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3361 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3421 gttccgcgca catttccccg aaaagtgcca c
//
