LOCUS       sequence-222724-        3319 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 3319)
  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 3319)
  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: pBoL1P3GA
FEATURES             Location/Qualifiers
     source          1..3319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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    392..413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(428..433,434..451,452..458)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    466..482
                     /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     471..493
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     490..506
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             502..825
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    519..575
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(576..592)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(801..820)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        922..997
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1009..1033)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1189..1206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1299..1887)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1379..1398)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2058..2849,2850..2918))
                     /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     2681..2700
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2919..3023)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgacgtaat acgactcact
       61 atagggcgaa ttggcggaag gccgtcaagg cctaggcgcg cccactctgt ggaagacaaa
      121 ctacatcttc attcttaaga tatgaagata atcttcaaaa ggcccctggg aatctgaaag
      181 aagagaagca ggcccattta tatgggaaag aacaatagta tttcttatat aggcccattt
      241 aagttgaaaa caatcttcaa aagtcccaca tcgcttagat aagaaaacga agctgagttt
      301 atatacagct agagtcgaag tagtgattgt gagaccgcag ctggcacgac aggtttcccg
      361 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      421 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      481 aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg
      541 actctagagg atccccgggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt
      601 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      661 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      721 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      781 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
      841 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      901 agacaagctg tgacggtctc agttttagag ctagaaatag caagttaaaa taaggctagt
      961 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctagac ccagctttct
     1021 tgtacaaagt tggcattacg ctttacttac aagtcttcca cgaagtgtta attaactggc
     1081 ctcatgggcc ttccgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1141 ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga
     1201 ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa
     1261 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1321 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1381 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1441 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1501 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1561 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1621 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1681 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1741 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1801 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1861 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1921 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1981 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2041 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2101 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2161 ggcttaccat ctggccccag tgctgcaatg ataccgcgag aaccacgctc accggctcca
     2221 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2281 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2341 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2401 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2461 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2521 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2581 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2641 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2701 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2761 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2821 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2881 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2941 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3001 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     3061 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3121 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3181 gccgctacag ggcgctccca ttcgccattc aggctgcgca actgttggga agggcgtttc
     3241 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     3301 aagttgggta acgccaggg
//
