LOCUS       sequence-413865-        3744 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 3744)
  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 3744)
  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: EC67848
FEATURES             Location/Qualifiers
     source          1..3744
                     /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           1033..1069
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    1153..1174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1189..1194,1195..1212,1213..1219)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1227..1243
                     /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     1232..1254
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1251..1267
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1263..1586
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1280..1336
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1337..1353)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1562..1581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1683..1750
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1887..1904)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1997..2585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2077..2096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2756..3547,3548..3616))
                     /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     3379..3398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3617..3721)
                     /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 aacagatctc acagagtgcc cctcatttat gtcaaactat
      421 acacttatca tgtctgacat gactttgcag ttaatttttt tgaacggtgt catgctttgc
      481 acaattgtcc tccatctata atgttttaca agaaactatt taatactgta ttaacctttg
      541 cagtaggcat acttggtacc ataattatct acatgctgtc atgtggagct cgtggtacat
      601 acagtagttt ttttgttact aaatacggag tacattcgta tatgatatgt tccctaatta
      661 gttgcatcac ttttttgtcc tcattgctgc tctactctaa acttatgttc cagacaagcc
      721 tacaactgag tcagcctgtc agagttctac tgtgtttgga tagatgttct agtgcaactt
      781 tcttcttgta tgtttagtta atcataagga ctcattttct tactacctcc gggacagatg
      841 tttctcagat gggccgggct ctctctgccc agcaggcagc agcagcccga cctcatgggc
      901 cagcacccac cacgagttcc agctaggggg tgttgctgcc taacactaac actaacatta
      961 gtcccacctc gccagtttgc agggatcgga accagcttat aagctgagct gaggaagaag
     1021 gtagcaaaat tactgatgag tccgtgagga cgaaacgagt aagctcgtct aatttctact
     1081 aagtgtagat tgagaccgca gctggcacga caggtttgcc gactggaaag cgggcagtga
     1141 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     1201 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     1261 ctatgaccat gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg
     1321 taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     1381 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     1441 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     1501 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     1561 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     1621 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacggtct
     1681 caggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg
     1741 cgaatgggac ttttttgtgt tgtcttcctg gcctgggcct catgggcctt ccgctcactg
     1801 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aacatggtca tagctgtttc
     1861 cttgcgtatt gggcgctctc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1921 gtaaagcctg gggtgcctaa tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2881 ctgcaatgat accgcgagaa ccacgctcac cggctccaga tttatcagca ataaaccagc
     2941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3721 gcacatttcc ccgaaaagtg ccac
//
