LOCUS       sequence-413832-        3450 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 3450)
  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 3450)
  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: EC70134
FEATURES             Location/Qualifiers
     source          1..3450
                     /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    905..926
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(941..946,947..964,965..971)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    979..995
                     /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     984..1006
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1003..1019
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1015..1338
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1032..1088
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1089..1105)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1314..1333)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1593..1610)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1703..2291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1783..1802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2462..3253,3254..3322))
                     /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     3085..3104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3323..3427)
                     /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 gcattctgtg aagacaatta cgaccaagcc 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 cttgccagtg gtctagtggt agaatagtac
      781 cctgccacgg tacagacccg ggttcgattc ccggctggaa ataatttcta ctaagtgtag
      841 attgagaccg cagctggcac gacaggtttg ccgactggaa agcgggcagt gagcgcaacg
      901 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
      961 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     1021 atgattacgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc
     1081 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     1141 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     1201 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     1261 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     1321 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     1381 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgacggt ctcataattt
     1441 ctactaagtg tagatttttt tcagattgtc ttcctggcct gggcctcatg ggccttccgc
     1501 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc
     1561 tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc
     1621 gttcgggtaa agcctggggt gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta
     1681 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1741 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1801 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1861 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1921 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     1981 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2041 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2101 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2161 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2221 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2281 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2341 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2401 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2461 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2521 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2581 ccagtgctgc aatgataccg cgagaaccac gctcaccggc tccagattta tcagcaataa
     2641 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2701 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2761 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2821 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2881 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     2941 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3001 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3061 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3121 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3181 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3241 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3301 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3361 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3421 ttccgcgcac atttccccga aaagtgccac
//
