LOCUS       sequence-413862-        3721 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 3721)
  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 3721)
  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: EC67898
FEATURES             Location/Qualifiers
     source          1..3721
                     /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    1176..1197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1212..1217,1218..1235,1236..1242)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1250..1266
                     /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     1255..1277
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1274..1290
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1286..1609
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1303..1359
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1360..1376)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1585..1604)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1864..1881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1974..2562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2054..2073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2733..3524,3525..3593))
                     /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     3356..3375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3594..3698)
                     /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 aagcaactca cagagtgccc ctcatttatg tcaaactata
      421 cacttatcat gtctgacatg actttgcagt taattttttt gaacggtgtc atgctttgca
      481 caattgtcct ccatctataa tgttttacaa gaaactattt aatactgtat taacctttgc
      541 agtaggcata cttggtacca taattatcta catgctgtca tgtggagctc gtggtacata
      601 cagtagtttt tttgttacta aatacggagt acattcgtat atgatatgtt ccctaattag
      661 ttgcatcact tttttgtcct cattgctgct ctactctaaa cttatgttcc agacaagcct
      721 acaactgagt cagcctgtca gagttctact gtgtttggat agatgttcta gtgcaacttt
      781 cttcttgtat gtttagttaa tcataaggac tcattttctt actacctccg ggacagatgt
      841 ttctcagatg ggccgggctc tctctgccca gcaggcagca gcagcccgac ctcatgggcc
      901 agcacccacc acgagttcca gctagggggt gttgctgcct aacactaaca ctaacattag
      961 tcccacctcg ccagtttgca gggatcggaa ccagcttata agctgagctg aggaagaagg
     1021 tagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt
     1081 cccggctgga aataatttct actaagtgta gattgagacc gcagctggca cgacaggttt
     1141 gccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     1201 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     1261 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc atgcctgcag
     1321 gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg ttttacaacg
     1381 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1441 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1501 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1561 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     1621 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     1681 ttacagacaa gctgtgacgg tctcataatt tctactaagt gtagattttt ttactattgt
     1741 cttcctggcc tgggcctcat gggccttccg ctcactgccc gctttccagt cgggaaacct
     1801 gtcgtgccag ctgcattaac atggtcatag ctgtttcctt gcgtattggg cgctctccgc
     1861 ttcctcgctc actgactcgc tgcgctcggt cgttcgggta aagcctgggg tgcctaatga
     1921 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1981 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2041 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2101 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2161 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2221 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2281 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2341 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2401 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2461 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2521 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2581 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2641 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2701 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2761 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2821 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaacca
     2881 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2941 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3001 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3061 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3121 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3181 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3241 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3301 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3361 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3421 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3481 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3541 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3601 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3661 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3721 c
//
