LOCUS       sequence-257083-        4446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for generating CRISPR arrays for composite array 
            utilized by SpCas9 and FnCas12a using CRATES. Contains a GFP-dropout
            cassette and two direct repeats. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4446)
  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 4446)
  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: pCas9FnCpf1GG
FEATURES             Location/Qualifiers
     source          1..4446
                     /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   75..110
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     RBS             270..281
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             288..1001
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1013..1084
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1100..1127
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1219..1236)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1389..1475
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1567..1594
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1613..1704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1705..1773,1774..2565)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSS
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1923..1942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2607..3062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2694..2713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2904..2925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3173..3761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3662..3681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3915..3932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3947..4087
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4013..4032)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4173..4195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4333..4352
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttgacagcta gctcagtcct aggtataatg ctagcgctag cgaattcgag ctcggtacca
       61 agacgtcaac tcgagtttta gagctatgct gttttgaatg gtcccaaaac ccctcgagac
      121 ggaaagtgaa acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt atttaataat
      181 gtgtgcggca attcacattt aatttatgaa tgttttctta acatcgcggc aactcaagaa
      241 acggcaggtt cggatcttag ctactagaga aagaggagaa atactagatg cgtaaaggcg
      301 aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc
      361 ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga
      421 agttcatctg tactactggt aaactgccgg ttccttggcc gactctggta acgacgctga
      481 cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca
      541 agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca
      601 cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc
      661 tgaaaggcat tgactttaaa gaggacggca atatcctggg ccataagctg gaatacaatt
      721 ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt
      781 ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac taccagcaaa
      841 acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg agcacgcaaa
      901 gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg gagttcgtaa
      961 ccgcagcggg catcacgcat ggtatggatg aactgtacaa atgaccaggc atcaaataaa
     1021 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1081 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacgtctct
     1141 aacggtctaa gaactttaaa taatttctac tgttgtagat aagcttggct gttttggcgg
     1201 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     1261 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     1321 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     1381 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1441 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     1501 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     1561 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     1621 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1681 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1741 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1801 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1861 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1921 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     1981 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2041 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2101 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2161 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2221 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2281 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2341 aaagttgcag gaccacttct gcgctcgtcc cttccggctg gctggtttat tgctgataaa
     2401 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2461 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2521 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2581 tactcatata tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg
     2641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2701 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2821 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2941 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3001 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3061 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3241 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3301 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3901 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3961 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     4021 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4081 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4141 ggcatccgct tacagacaag ctgtgacggt ctccgggagc tgcatgtgtc agaggttttc
     4201 accgtcatca ccgaaacgcg cgaggcagaa ggagatggcg cccaacagtc ccccggccac
     4261 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     4321 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     4381 gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag cttatcatcg
     4441 atgcat
//
