LOCUS       sequence-413839-        3602 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 3602)
  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 3602)
  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: EC70129
FEATURES             Location/Qualifiers
     source          1..3602
                     /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(1745..1762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1855..2443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1935..1954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2614..3405,3406..3474))
                     /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     3237..3256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3475..3579)
                     /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 aaactacatg 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 attttttttt
     1621 acttgtcttc ctgggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc
     1681 tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg gcgctctccg
     1741 cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg gtgcctaatg
     1801 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1861 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1921 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1981 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2041 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2101 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2161 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2221 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2281 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2341 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2401 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2461 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2521 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2581 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2641 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2701 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc
     2761 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2821 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2881 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2941 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3001 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3061 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3121 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3181 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3241 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3301 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3361 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3421 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3481 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3541 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3601 ac
//
