LOCUS       sequence-310179-        4141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for LbCas12a crRNAs ( Step 1 of the 2-step cloning 
            alternative). Full PgpdA sequence is reconstituted when amplifying 
            the expression cassette for cloning in a fungal vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4141)
  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 4141)
  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: pCRI007-pGEM-PgpdA-Cas12aScaffold-BsmbI-TtrpC
FEATURES             Location/Qualifiers
     source          1..4141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        133..151
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     133..150
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(969..985)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(970..986)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     regulatory      1371..1380
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1375..1384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1414..1433)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1415..1433)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1440..1457)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1440..1456)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1449..1471)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1597..2052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1684..1703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1894..1915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2130..2234
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2235..2303,2304..3095)
                     /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(2453..2472)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3266..3854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3755..3774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4008..4025
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatcc aacgcgttgg
      181 gagctctccc atatggtcga cctgcaggcg gccgcgaatt cctcgtcgtg tcgcccatta
      241 attaagacgg acggtcgcag agagaagggc tgagtaataa gcgccactgc gccagacagc
      301 tctggcggct ctgaggtgca gtggatgatt attaatccgg gaccggccgc ccctccgccc
      361 cgaagtggaa aggctggtgt gcccctcgtt gaccaagaat ctattgcatc atcggagaat
      421 atggagcttc atcgaatcac cggcagtaag cgaaggagaa tgtgaagcca ggggtgtata
      481 gccgtcggcg aaatagcatg ccattaacct aggtacagaa gtccaattgc ttccgatctg
      541 gtaaaagatt cacgagatag taccttctcc gaagtaggta gagcgagtac ccggcgcgta
      601 agctccctaa ttggcccatc cggcatctgt agggcgtcca aatatcgtgc ctctcctgct
      661 ttgcccggtg tatgaaaccg gaaaggccgc tcaggagctg gccagcggcg cagaccggga
      721 acacaagctg gcagtcgacc catccggtgc tctgcactcg acctgctgag gtccctcagt
      781 ccctggtagg cagctttgcc ccgtctgtcc gcccggtgtg tcggcggggt tgacaaggtc
      841 gttgcgtcag tccaacattt gttgccatat tttcctgctc tccccaccag ctgctctttt
      901 cttttctctt tcttttccca tcttcagtat attcatcttc ccatccaaga acctttaatc
      961 aagcttatcg ataccgtcga cctcgactct agaggatcga atttctacta agtgtagatg
     1021 gagacgagaa ttccgtctcc aatttctact aagtgtagat atcttcgagg gggggcccgg
     1081 taccgccccg tccggtcctg cccgtcaccg agatccactt aacgttactg aaatcatcaa
     1141 acagcttgac gaatctggat ataagatcgt tggtgtcgat gtcagctccg gagttgagac
     1201 aaatggtgtt caggatctcg ataagatacg ttcatttgtc caagcagcaa agagtgcctt
     1261 ctagtgattt aatagctcca tgtcaacaag aataaaacgc gtttcgggtt tacctcttcc
     1321 agatacagct catctgcaat gcatgggcga cgacgacgtg aattcccgcg gccgccatgg
     1381 cggccgggag catgcgacgt cgggcccaat tcgccctata gtgagtcgta ttacaattca
     1441 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     1501 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     1561 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     1621 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1681 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1741 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1801 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1861 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1921 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1981 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2041 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2101 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2161 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2221 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2281 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2341 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2401 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2461 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2521 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2581 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2641 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2701 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2761 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2821 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2881 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2941 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3001 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3061 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3121 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3181 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3241 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3301 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3361 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3421 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3481 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3541 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3601 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3661 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3721 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3781 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3841 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3901 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3961 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4021 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4081 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4141 t
//
