LOCUS       sequence-413845-        3743 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 3743)
  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 3743)
  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: EC67845
FEATURES             Location/Qualifiers
     source          1..3743
                     /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           1032..1068
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    1152..1173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1188..1193,1194..1211,1212..1218)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1226..1242
                     /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     1231..1253
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1250..1266
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1262..1585
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1279..1335
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1336..1352)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1561..1580)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1682..1749
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1886..1903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1996..2584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2076..2095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2755..3546,3547..3615))
                     /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     3378..3397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3616..3720)
                     /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 tagcaaaatt actgatgagt ccgtgaggac gaaacgagta agctcgtcta atttctacta
     1081 agtgtagatt gagaccgcag ctggcacgac aggtttgccg actggaaagc gggcagtgag
     1141 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     1201 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     1261 tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt
     1321 accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1381 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1441 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     1501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     1561 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     1621 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgacggtctc
     1681 aggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacatgct tcggcatggc
     1741 gaatgggact ttttactatt gtcttcctgg cctgggcctc atgggccttc cgctcactgc
     1801 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat agctgtttcc
     1861 ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggg
     1921 taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1981 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2041 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2101 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2161 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2221 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2281 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2341 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2401 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2461 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2521 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2581 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2641 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2701 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2761 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2821 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     2881 tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     2941 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3001 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3061 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3121 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3181 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3241 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3301 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3361 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3421 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3481 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3541 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3601 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3661 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3721 cacatttccc cgaaaagtgc cac
//
