LOCUS       sequence-413835-        3612 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 3612)
  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 3612)
  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: EC70132
FEATURES             Location/Qualifiers
     source          1..3612
                     /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    1067..1088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1103..1108,1109..1126,1127..1133)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1141..1157
                     /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     1146..1168
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1165..1181
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1177..1500
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1194..1250
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1251..1267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1476..1495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1755..1772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1865..2453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1945..1964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2624..3415,3416..3484))
                     /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     3247..3266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3485..3589)
                     /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 aagacaatgt gcatgaatcc aaaccacacg gagttcaaat
      421 tcccacagat taaggctcgt ccgtcgcaca aggtaatgtg tgaatattat atctgtcgtg
      481 caaaattgcc tggcctgcac aattgctgtt atagttggcg gcagggagag ttttaacatt
      541 gactagcgtg ctgataattt gtgagaaata ataattgaca agtagatact gacatttgag
      601 aagagcttct gaactgttat tagtaacaaa aatggaaagc tgatgcacgg aaaaaggaaa
      661 gaaaaagcca tacttttttt taggtaggaa aagaaaaagc catacgagac tgatgtctct
      721 cagatgggcc gggatctgtc tatctagcag gcagcagccc accaacctca cgggccagca
      781 attacgagtc cttctaaaag ctcccgccga ggggcgctgg cgctgctgtg cagcagcacg
      841 tctaacatta gtcccacctc gccagtttac agggagcaga accagcttat aagcggaggc
      901 gcggcaccaa gaagcaccag tggtctagtg gtagaatagt accctgccac ggtacagacc
      961 cgggttcgat tcccggctgg aaataatttc tactaagtgt agattgagac cgcagctggc
     1021 acgacaggtt tgccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     1081 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     1141 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
     1201 catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc actggccgtc
     1261 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     1321 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     1381 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     1441 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     1501 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     1561 ccggcatccg cttacagaca agctgtgacg gtctcataat ttctactaag tgtagatttt
     1621 tttgagcttg tcttcctggc ctgggcctca tgggccttcc gctcactgcc cgctttccag
     1681 tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg
     1741 gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg
     1801 gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2701 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2761 cgcgagaacc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2821 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2881 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2941 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3001 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3061 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3121 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3181 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3241 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3301 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3361 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3541 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3601 gaaaagtgcc ac
//
