LOCUS       sequence-222715-        3789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a cloned sgRNA from AtU626 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3789)
  AUTHORS   Lawrenson T, Hundleby P, Harwood W
  TITLE     Creating Targeted Gene Knockouts in Brassica oleracea Using 
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1917:155-170. doi: 
            10.1007/978-1-4939-8991-1_12.
  PUBMED    30610635
REFERENCE   2  (bases 1 to 3789)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1917:155-170. doi"; pages: " 
            10.1007/978-1-4939-8991-1_12"
COMMENT     Sequence Label: pBoL1P4GA
FEATURES             Location/Qualifiers
     source          1..3789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     19..36
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     20..36
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     47..66
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        47..65
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    427..448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(463..468,469..486,487..493)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    501..517
                     /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)."
     RBS             535..546
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             553..1230
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1275..1346
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1362..1389
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(1486..1510)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1659..1676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1769..2357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1849..1868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2528..3319,3320..3388))
                     /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     3151..3170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3389..3493)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgacgtaat acgactcact
       61 atagggcgaa ttggcggaag gccgtcaagg ccgcatcact ctgtggaaga caattaccat
      121 cttcattctt aagatatgaa gataatcttc aaaaggcccc tgggaatctg aaagaagaga
      181 agcaggccca tttatatggg aaagaacaat agtatttctt atataggccc atttaagttg
      241 aaaacaatct tcaaaagtcc cacatcgctt agataagaaa acgaagctga gtttatatac
      301 agctagagtc gaagtagtgc gagacgcaat acgcaaaccg cctctccccg cgcgttggcc
      361 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      421 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      481 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacatact agagaaagag
      541 gagaaatact agatggcttc ctccgaggac gttatcaaag agttcatgcg tttcaaagtt
      601 cgtatggaag gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt
      661 ccgtacgaag gtacccagac cgctaaactg aaagttacca aaggtggtcc gctgccgttc
      721 gcttgggaca tcctgtcccc gcagttccag tacggttcca aagcttacgt taaacacccg
      781 gctgacatcc cggactacct gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt
      841 atgaacttcg aggacggtgg tgttgttacc gttacccagg actcctccct gcaagacggt
      901 gagttcatct acaaagttaa actgcgtggt accaacttcc cgtccgacgg tccggttatg
      961 cagaaaaaaa ccatgggttg ggaagcttcc accgaacgta tgtacccgga ggacggtgct
     1021 ctgaaaggtg aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa
     1081 gttaaaacca cctacatggc taaaaaaccg gttcagctgc cgggtgctta caaaaccgac
     1141 atcaaactgg acatcacctc ccacaacgag gactacacca tcgttgaaca gtacgaacgt
     1201 gctgaaggtc gtcactccac cggtgcttaa taacgctgat agtgctagtg tagatcgcta
     1261 ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     1321 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     1381 ctttctgcgt ttatacgtct cttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1441 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt
     1501 acaaagttgg cattacgctt tacgggaaag tcttccacga agtgctgggc ctcatgggcc
     1561 ttccgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaacatggt
     1621 catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga ctcgctgcgc
     1681 tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa aaggccagga
     1741 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1801 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1861 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1921 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1981 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2041 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2101 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2161 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2221 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2281 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2341 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2401 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2461 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2521 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2581 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2641 ctggccccag tgctgcaatg ataccgcgag aaccacgctc accggctcca gatttatcag
     2701 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2761 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2821 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2881 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2941 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3001 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3061 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3121 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3181 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3241 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3301 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3361 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3481 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     3541 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3601 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg gccgctacag
     3661 ggcgctccca ttcgccattc aggctgcgca actgttggga agggcgtttc ggtgcgggcc
     3721 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     3781 acgccaggg
//
