LOCUS       sequence-413980-        3473 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 3473)
  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 3473)
  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: EC70128
FEATURES             Location/Qualifiers
     source          1..3473
                     /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           762..798
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    882..903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(918..923,924..941,942..948)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    956..972
                     /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     961..983
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     980..996
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             992..1315
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1009..1065
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1066..1082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1291..1310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1412..1479
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1616..1633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1726..2314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1806..1825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2485..3276,3277..3345))
                     /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     3108..3127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3346..3450)
                     /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 aggccgcata 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 acttgaaatt actgatgagt ccgtgaggac
      781 gaaacgagta agctcgtcta atttctacta agtgtagatt gagaccgcag ctggcacgac
      841 aggtttgccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      901 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      961 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc
     1021 ctgcaggtcg actctagagg atccccgggt accgagctcg aattcactgg ccgtcgtttt
     1081 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1141 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1201 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     1261 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1321 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1381 atccgcttac agacaagctg tgacggtctc aggccggcat ggtcccagcc tcctcgctgg
     1441 cgccggctgg gcaacatgct tcggcatggc gaatgggact ttttgagctt gtcttcctgg
     1501 cctgggcctc atgggccttc cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1561 agctgcatta acatggtcat agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc
     1621 tcactgactc gctgcgctcg gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg
     1681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     1741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     1801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1921 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2161 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     2341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     2401 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2461 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2521 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2581 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc
     2641 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2701 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2761 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2821 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2881 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2941 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3001 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3061 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3121 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3181 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3241 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3301 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
