LOCUS       sequence-310772-        3711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for cloning Cas9 sgRNA ( Step 1 of 2-step cloning). Contains 
            AfU3 promoter driven sgRNA expression cassette flanked by PacI and 
            NotI for further cloning in fungal vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3711)
  AUTHORS   Roux I, Woodcraft C, Hu J, Wolters R, Gilchrist CLM, Chooi YH
  TITLE     CRISPR-mediated activation of biosynthetic gene clusters for 
            bioactive molecule discovery in filamentous fungi.
  JOURNAL   ACS Synth Biol. 2020 Jun 11. doi: 10.1021/acssynbio.0c00197.
  PUBMED    32526136
REFERENCE   2  (bases 1 to 3711)
  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: "ACS Synth 
            Biol. 2020 Jun 11. doi: 10.1021/acssynbio.0c00197."
COMMENT     Sequence Label: pCRI012-pGEM-PacI-PU3-BsmbI-NotI
FEATURES             Location/Qualifiers
     source          1..3711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      73..82
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      77..86
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        722..797
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(876..894)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(877..894)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(912..928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(912..928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(925..947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    936..952
                     /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)."
     promoter        complement(join(960..966,967..984,985..990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1005..1026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1143..1160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1314..1902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1394..1413)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2073..2864,2865..2933))
                     /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     2696..2715
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2934..3038)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3116..3571)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3253..3274)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3465..3484
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(3697..3711,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3711,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 cgcatgctcc cggccgccat ggcggccgcg aattcctcgt cgtgtcgccc attaattaag
      121 atcacataga tgctcggttg acaggaccac ctatagcaag tactttgtag gaggttctcc
      181 taacgcttgg cttcaaatcc aatcaggata tcagtgagtt actgtttgtc atcgcatcct
      241 catgaagtcg ctcaacagcc actgagaagc aaatatttgg gagaccatcc ctgatgttga
      301 aattttacgc tggagcccat tcaccggtga gctcggggac agtctggcca gtggagcgga
      361 aaatcttcaa ctaactctga ttggtaccac aggtagcccc acagaacaaa ccgagcaaac
      421 atgaaaattt tcgcttgagg ttagcgcact cgctagcgcc tgcccgcaaa tataaaaggc
      481 cccgaaatcc catgcatttt ggaaatcaag cgactctacg tatgtcatcc ccctccctac
      541 ctttgggaaa acctctcaac tgcaagtcag aacattttgc taacagcaga gacgggcgcc
      601 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
      661 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgcgtctc
      721 cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      781 tggcaccgag tcggtgcttt tttttcaatt cgcggccgcc tgcaggtcga ccatatggga
      841 gagctcccaa cgcgttggat gcatagcttg agtattctat agtgtcacct aaatagcttg
      901 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      961 aacatacgag ccggaagcat aaagtgcaaa gcctggggtg cctaatgagt gagctaactc
     1021 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1081 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1141 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1201 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1261 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1321 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1381 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1441 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1501 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1561 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1621 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1681 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1741 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1801 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1861 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1921 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1981 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2041 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2101 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2161 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2221 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2281 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2341 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2401 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2461 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2521 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2581 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2641 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2701 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2761 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2821 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2881 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2941 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3001 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3061 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     3121 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3181 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3241 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3301 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3361 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3421 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3481 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3541 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     3601 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     3661 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt t
//
