LOCUS       sequence-257082-        4286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for generating CRISPR arrays for SpCas9 using 
            CRATES. Contains a direct repeat and a RFP-dropout cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4286)
  AUTHORS   Liao C, Ttofali F, Slotkowski RA, Denny SR, Cecil TD, Leenay RT, 
            Keung AJ, Beisel CL
  TITLE     Modular one-pot assembly of CRISPR arrays enables library generation
            and reveals factors influencing crRNA biogenesis.
  JOURNAL   Nat Commun. 2019 Jul 3;10(1):2948. doi: 10.1038/s41467-019-10747-3.
  PUBMED    31270316
REFERENCE   2  (bases 1 to 4286)
  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: "Nat 
            Commun."; date: "2019-07-3"; volume: "10(1):2948. doi"; pages: " 
            10.1038/s41467-019-10747-3"
COMMENT     Sequence Label: pCas9GG
FEATURES             Location/Qualifiers
     source          1..4286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     repeat_region   68..103
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    115..133
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    140..158
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             176..187
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             194..871
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      889..960
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      976..1003
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1059..1076)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1229..1315
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1407..1434
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1453..1544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1545..1613,1614..2405)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1763..1782)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2447..2902
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2534..2553)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2744..2765
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3013..3601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3502..3521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3755..3772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3787..3927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3853..3872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4013..4035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4173..4192
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttgacagcta gctcagtcct aggtataatg ctagcgctag cgaattcgag ctcggtacca
       61 agacgtcgtt ttagagctat gctgttttga atggtcccaa aaccgcagga gacgtcccta
      121 tcagtgatag agattgacat ccctatcagt gatagagata ctgagcacgg atctgaaaga
      181 ggagaaagga tctatggcga gtagcgaaga cgttatcaaa gagttcatgc gtttcaaagt
      241 tcgtatggaa ggttccgtta acggtcacga gttcgaaatc gaaggtgaag gtgaaggtcg
      301 tccgtacgaa ggtactcaga ccgctaaact gaaagttacc aaaggtggtc cgctgccgtt
      361 cgcttgggac atcctgtccc cgcagttcca gtacggttcc aaagcttacg ttaaacaccc
      421 ggctgacatc ccggactacc tgaaactgtc cttcccggaa ggtttcaaat gggaacgtgt
      481 tatgaacttc gaagacggtg gtgttgttac cgttacccag gactcctccc tgcaagacgg
      541 tgagttcatc tacaaagtta aactgcgtgg tactaacttc ccgtccgacg gtccggttat
      601 gcagaaaaaa accatgggtt gggaagcttc caccgaacgt atgtacccgg aagacggtgc
      661 tctgaaaggt gaaatcaaaa tgcgtctgaa actgaaagac ggtggtcact acgacgctga
      721 agttaaaacc acctacatgg ctaaaaaacc ggttcagctg ccgggtgctt acaaaaccga
      781 catcaaactg gacatcacct cccacaacga agactacacc atcgttgaac agtacgaacg
      841 tgctgaaggt cgtcactcca ccggtgctta ataaggatct ccaggcatca aataaaacga
      901 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
      961 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac gtctcattgc
     1021 aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     1081 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     1141 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     1201 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     1261 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     1321 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     1381 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     1441 tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1501 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1561 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1621 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1681 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1741 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     1801 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1861 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1921 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1981 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2041 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2101 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2161 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2221 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2281 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2341 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2401 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttacgc gccctgtagc
     2461 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2521 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2581 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2641 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     2701 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2761 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     2821 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2881 aaaatattaa cgtttacaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3121 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3781 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     3841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3901 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3961 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgacggt ctccgggagc
     4021 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagaa ggagatggcg
     4081 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4141 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     4201 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca
     4261 tgtttgacag cttatcatcg atgcat
//
