LOCUS       sequence-413850-        3440 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 3440)
  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 3440)
  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: EC70133
FEATURES             Location/Qualifiers
     source          1..3440
                     /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    900..921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(936..941,942..959,960..966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    974..990
                     /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     979..1001
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     998..1014
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1010..1333
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1027..1083
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1084..1100)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1309..1328)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1583..1600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1693..2281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1773..1792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2452..3243,3244..3312))
                     /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     3075..3094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3313..3417)
                     /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 aggccgcatt ctgtgaagac aaactagacc aagcccgtta ttctgacagt tctggtgctc
      421 aacacattta tatttatcaa ggagcacatt gttactcact gctaggaggg aatcgaacta
      481 ggaatattga tcagaggaac tacgagagag ctgaagataa ctgccctcta gctctcactg
      541 atctgggtcg catagtgaga tgcagcccac gtgagttcag caacggtcta gcgctgggct
      601 tttaggcccg catgatcggg cttttgtcgg gtggtcgacg tgttcacgat tggggagagc
      661 aacgcagcag ttcctcttag tttagtccca cctcgcctgt ccagcagagt tctgaccggt
      721 ttataaactc gcttgctgca tcagacttgc cagtggtcta gtggtagaat agtaccctgc
      781 cacggtacag acccgggttc gattcccggc tggaaataat ttctactaag tgtagattga
      841 gaccgcagct ggcacgacag gtttgccgac tggaaagcgg gcagtgagcg caacgcaatt
      901 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      961 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1021 tacgccaagc ttgcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa
     1081 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1141 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     1201 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     1261 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     1321 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     1381 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg acggtctcat aatttctact
     1441 aagtgtagat ttttttttac ttgtcttcct gggcctcatg ggccttccgc tcactgcccg
     1501 ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc tgtttccttg
     1561 cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc gttcgggtaa
     1621 agcctggggt gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1681 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1741 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1801 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1861 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1921 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1981 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2041 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2101 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2161 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2221 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2281 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2341 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     2401 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2461 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2521 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2581 aatgataccg cgagaaccac gctcaccggc tccagattta tcagcaataa accagccagc
     2641 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2701 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2761 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2821 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2881 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2941 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3001 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3061 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3121 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3181 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     3241 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     3301 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     3361 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3421 atttccccga aaagtgccac
//
