LOCUS       sequence-252009-        4287 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 4287)
  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 4287)
  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..4287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      288..876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     777..796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1030..1047
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1062..1202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1128..1147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1288..1310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1449..1468
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1563..1597
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     repeat_region   1630..1665
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     protein_bind    1677..1695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1702..1720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1738..1749
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1756..2433
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2451..2522
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2538..2565
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2621..2638)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2791..2877
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2969..2996
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3015..3106
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3107..3175,3176..3967)
                     /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(3325..3344)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4009..4287,1..177)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4096..4115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 agtccacgtt ctttaatagt ggactcttgt tccaaacttg aacaacactc aaccctatct
       61 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      121 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa
      181 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
      241 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      301 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      361 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
      421 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      481 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
      541 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      601 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
      661 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      721 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      781 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      841 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      901 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      961 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1021 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     1081 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     1141 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     1201 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     1261 ccgcttacag acaagctgtg acggtctccg ggagctgcat gtgtcagagg ttttcaccgt
     1321 catcaccgaa acgcgcgagg cagcaaggag atggcgccca acagtccccc ggccacgggg
     1381 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     1441 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     1501 ccggccacga tgcgtccggc gtagaggatc tgctcatgtt tgacagctta tcatcgatgc
     1561 atttgacagc tagctcagtc ctaggtataa tgctagcgct agcgaattcg agctcggtac
     1621 caagacgtcg ttttagagct atgctgtttt gaatggtccc aaaaccgcag gagacgtccc
     1681 tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac ggatctgaaa
     1741 gaggagaaag gatctatggc gagtagcgaa gacgttatca aagagttcat gcgtttcaaa
     1801 gttcgtatgg aaggttccgt taacggtcac gagttcgaaa tcgaaggtga aggtgaaggt
     1861 cgtccgtacg aaggtactca gaccgctaaa ctgaaagtta ccaaaggtgg tccgctgccg
     1921 ttcgcttggg acatcctgtc cccgcagttc cagtacggtt ccaaagctta cgttaaacac
     1981 ccggctgaca tcccggacta cctgaaactg tccttcccgg aaggtttcaa atgggaacgt
     2041 gttatgaact tcgaagacgg tggtgttgtt accgttaccc aggactcctc cctgcaagac
     2101 ggtgagttca tctacaaagt taaactgcgt ggtactaact tcccgtccga cggtccggtt
     2161 atgcagaaaa aaaccatggg ttgggaagct tccaccgaac gtatgtaccc ggaagacggt
     2221 gctctgaaag gtgaaatcaa aatgcgtctg aaactgaaag acggtggtca ctacgacgct
     2281 gaagttaaaa ccacctacat ggctaaaaaa ccggttcagc tgccgggtgc ttacaaaacc
     2341 gacatcaaac tggacatcac ctcccacaac gaagactaca ccatcgttga acagtacgaa
     2401 cgtgctgaag gtcgtcactc caccggtgct taataaggat ctccaggcat caaataaaac
     2461 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2521 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acgtctcatt
     2581 gcaagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     2641 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     2701 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     2761 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     2821 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     2881 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     2941 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     3001 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3061 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3121 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3181 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3241 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3301 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     3361 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3421 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3481 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3541 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3601 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     3661 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3721 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3781 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3841 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3901 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3961 ttggtaactg tcagaccaag tttactcata tatactttag attgatttac gcgccctgta
     4021 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4081 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4141 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4201 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4261 agacggtttt tcgccctttg acgttgg
//
