LOCUS       sequence-419151-        3341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of sgRNAs for expression in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3341)
  TITLE     Christian Staehelin Lab Plasmids 2023
REFERENCE   2  (bases 1 to 3341)
  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"
COMMENT     Sequence Label: pUC-tracrRNA-PMtU6-26
FEATURES             Location/Qualifiers
     source          1..3341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(695..712)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(695..711)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(704..726)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(920..939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1039..1061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1099..1117)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1185..1289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1290..1358,1359..2150)
                     /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     complement(1508..1527)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2321..2909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2810..2829
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3063..3080
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3197..3218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3233..3238,3239..3256,3257..3263)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3271..3287
                     /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     3276..3298
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3295..3311
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3295..3311
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat
      121 ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gataggaaat tttgctggga
      181 aacaaattta ctcgaaatat caaattgccc ccagaaactt aaagttaaaa aattaaaatt
      241 cctcaagatt ttctgaggga attttcatgg aaaacaaatg tatttggatt gctcgcttgc
      301 aatccgagga atatatgctt ggaatatttt catctgctct tccatgggct ctatatgatg
      361 cattgatatc cttcaactta tatggcttag tgtgaaaaaa aatagtgtaa ctagggaagg
      421 gtaacagatt gtgaatgaat tggagaagct aaagatggaa cgagcagatg aagttaaata
      481 agtgatatac ttgcggtgga gcaatgcatg gttaaggcag gatatgatga ggcaatatag
      541 tcatccagaa aaagcatcgg cgggcgagac tgagcatata ccaattactg gagtcccaca
      601 tcgcttaaat gttatgcatc ttcagtgttt atatatcgtc acagagcaca atggcttatt
      661 gagagacctc tgaagataac atactaagct tggcactggc cgtcgtttta caacgtcgtg
      721 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      781 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      841 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
      901 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
      961 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1021 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     1081 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     1141 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1201 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1261 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1321 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1381 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1441 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1501 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1561 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1621 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1681 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1741 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1801 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1861 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1921 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1981 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2041 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2101 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2161 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2221 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2281 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2341 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2401 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2461 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2521 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2581 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2641 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2701 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2761 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2821 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2881 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2941 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3001 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3061 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3121 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3181 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     3241 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     3301 acagctatga ccatgattac gaattcgagc tcggtacccg g
//
