LOCUS       sequence-413859-        3711 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 3711)
  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 3711)
  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: EC67899
FEATURES             Location/Qualifiers
     source          1..3711
                     /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    1171..1192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1207..1212,1213..1230,1231..1237)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1245..1261
                     /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     1250..1272
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1269..1285
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1281..1604
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1298..1354
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1355..1371)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1580..1599)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1854..1871)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1964..2552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2044..2063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2723..3514,3515..3583))
                     /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     3346..3365
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3584..3688)
                     /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 actcacagag tgcccctcat ttatgtcaaa ctatacactt
      421 atcatgtctg acatgacttt gcagttaatt tttttgaacg gtgtcatgct ttgcacaatt
      481 gtcctccatc tataatgttt tacaagaaac tatttaatac tgtattaacc tttgcagtag
      541 gcatacttgg taccataatt atctacatgc tgtcatgtgg agctcgtggt acatacagta
      601 gtttttttgt tactaaatac ggagtacatt cgtatatgat atgttcccta attagttgca
      661 tcactttttt gtcctcattg ctgctctact ctaaacttat gttccagaca agcctacaac
      721 tgagtcagcc tgtcagagtt ctactgtgtt tggatagatg ttctagtgca actttcttct
      781 tgtatgttta gttaatcata aggactcatt ttcttactac ctccgggaca gatgtttctc
      841 agatgggccg ggctctctct gcccagcagg cagcagcagc ccgacctcat gggccagcac
      901 ccaccacgag ttccagctag ggggtgttgc tgcctaacac taacactaac attagtccca
      961 cctcgccagt ttgcagggat cggaaccagc ttataagctg agctgaggaa gaaggtagca
     1021 ccagtggtct agtggtagaa tagtaccctg ccacggtaca gacccgggtt cgattcccgg
     1081 ctggaaataa tttctactaa gtgtagattg agaccgcagc tggcacgaca ggtttgccga
     1141 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     1201 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     1261 atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga
     1321 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
     1381 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1441 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     1501 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     1561 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     1621 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1681 gacaagctgt gacggtctca taatttctac taagtgtaga ttttttttta cttgtcttcc
     1741 tgggcctcat gggccttccg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1801 ctgcattaac atggtcatag ctgtttcctt gcgtattggg cgctctccgc ttcctcgctc
     1861 actgactcgc tgcgctcggt cgttcgggta aagcctgggg tgcctaatga gcaaaaggcc
     1921 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1981 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2041 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2101 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2161 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2221 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2281 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2341 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2401 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2461 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2521 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2581 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2641 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2701 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2761 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2821 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaacca cgctcaccgg
     2881 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2941 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3001 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3061 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3121 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3181 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3241 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3301 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3361 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3421 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3481 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3541 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3601 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3661 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
