LOCUS       sequence-413866-        3472 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 3472)
  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 3472)
  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: EC70125
FEATURES             Location/Qualifiers
     source          1..3472
                     /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           761..797
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     protein_bind    881..902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(917..922,923..940,941..947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    955..971
                     /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     960..982
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     979..995
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             991..1314
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1008..1064
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1065..1081)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1290..1309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     ncRNA           1411..1478
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1615..1632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1725..2313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1805..1824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2484..3275,3276..3344))
                     /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     3107..3126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3345..3449)
                     /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 aagacaaact agaccaagcc cgttattctg acagttctgg
      421 tgctcaacac atttatattt atcaaggagc acattgttac tcactgctag gagggaatcg
      481 aactaggaat attgatcaga ggaactacga gagagctgaa gataactgcc ctctagctct
      541 cactgatctg ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg gtctagcgct
      601 gggcttttag gcccgcatga tcgggctttt gtcgggtggt cgacgtgttc acgattgggg
      661 agagcaacgc agcagttcct cttagtttag tcccacctcg cctgtccagc agagttctga
      721 ccggtttata aactcgcttg ctgcatcaga cttgaaatta ctgatgagtc cgtgaggacg
      781 aaacgagtaa gctcgtctaa tttctactaa gtgtagattg agaccgcagc tggcacgaca
      841 ggtttgccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      901 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      961 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc
     1021 tgcaggtcga ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta
     1081 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     1141 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     1201 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1261 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     1321 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     1381 tccgcttaca gacaagctgt gacggtctca ggccggcatg gtcccagcct cctcgctggc
     1441 gccggctggg caacatgctt cggcatggcg aatgggactt tttttacttg tcttcctggc
     1501 ctgggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1561 gctgcattaa catggtcata gctgtttcct tgcgtattgg gcgctctccg cttcctcgct
     1621 cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg gtgcctaatg agcaaaaggc
     1681 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1741 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1801 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1861 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1921 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1981 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2041 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2101 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2161 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2221 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2281 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2341 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2401 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2461 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2521 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2581 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg
     2641 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2701 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2761 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2821 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2881 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2941 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3001 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3061 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3121 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3181 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3241 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3301 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3361 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3421 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
