LOCUS       sequence-413825-        3607 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 3607)
  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 3607)
  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: EC67892
FEATURES             Location/Qualifiers
     source          1..3607
                     /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    1062..1083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1098..1103,1104..1121,1122..1128)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1136..1152
                     /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     1141..1163
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1160..1176
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1172..1495
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1189..1245
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1246..1262)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1471..1490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1750..1767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1860..2448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1940..1959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2619..3410,3411..3479))
                     /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     3242..3261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3480..3584)
                     /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 aagcaacatg aatccaaacc acacggagtt caaattccca
      421 cagattaagg ctcgtccgtc gcacaaggta atgtgtgaat attatatctg tcgtgcaaaa
      481 ttgcctggcc tgcacaattg ctgttatagt tggcggcagg gagagtttta acattgacta
      541 gcgtgctgat aatttgtgag aaataataat tgacaagtag atactgacat ttgagaagag
      601 cttctgaact gttattagta acaaaaatgg aaagctgatg cacggaaaaa ggaaagaaaa
      661 agccatactt ttttttaggt aggaaaagaa aaagccatac gagactgatg tctctcagat
      721 gggccgggat ctgtctatct agcaggcagc agcccaccaa cctcacgggc cagcaattac
      781 gagtccttct aaaagctccc gccgaggggc gctggcgctg ctgtgcagca gcacgtctaa
      841 cattagtccc acctcgccag tttacaggga gcagaaccag cttataagcg gaggcgcggc
      901 accaagaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt
      961 tcgattcccg gctggaaata atttctacta agtgtagatt gagaccgcag ctggcacgac
     1021 aggtttgccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     1081 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     1141 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc
     1201 ctgcaggtcg actctagagg atccccgggt accgagctcg aattcactgg ccgtcgtttt
     1261 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1321 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1381 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     1441 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1501 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1561 atccgcttac agacaagctg tgacggtctc ataatttcta ctaagtgtag atttttttac
     1621 tattgtcttc ctggcctggg cctcatgggc cttccgctca ctgcccgctt tccagtcggg
     1681 aaacctgtcg tgccagctgc attaacatgg tcatagctgt ttccttgcgt attgggcgct
     1741 ctccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cgggtaaagc ctggggtgcc
     1801 taatgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1861 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1921 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1981 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2041 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2101 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2161 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2221 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2281 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2341 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2401 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2461 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2521 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2581 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2641 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2701 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2761 gaaccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2821 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2881 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2941 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3001 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3061 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3121 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3181 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3241 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3301 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3361 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3421 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3481 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3541 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3601 gtgccac
//
