LOCUS       sequence-252342-        4447 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 4447)
  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 4447)
  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..4447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     166..185
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        280..314
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     repeat_region   354..389
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     RBS             549..560
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             567..1280
                     /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      1292..1363
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1379..1406
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1498..1515)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1668..1754
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1846..1873
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1892..1983
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1984..2052,2053..2844)
                     /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(2202..2221)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2886..3341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2973..2992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3452..4040
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3941..3960
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4194..4211
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4226..4366
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4292..4311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
       61 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
      121 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      181 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      241 gaggatctgc tcatgtttga cagcttatca tcgatgcatt tgacagctag ctcagtccta
      301 ggtataatgc tagcgctagc gaattcgagc tcggtaccaa gacgtcaact cgagttttag
      361 agctatgctg ttttgaatgg tcccaaaacc cctcgagacg gaaagtgaaa cgtgatttca
      421 tgcgtcattt tgaacatttt gtaaatctta tttaataatg tgtgcggcaa ttcacattta
      481 atttatgaat gttttcttaa catcgcggca actcaagaaa cggcaggttc ggatcttagc
      541 tactagagaa agaggagaaa tactagatgc gtaaaggcga agagctgttc actggtgtcg
      601 tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg
      661 agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta
      721 aactgccggt tccttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg
      781 ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct
      841 atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag
      901 tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag
      961 aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca
     1021 tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg
     1081 aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc
     1141 ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga
     1201 acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg
     1261 gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     1321 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
     1381 ctcaccttcg ggtgggcctt tctgcgttta tacgtctcta acggtctaag aactttaaat
     1441 aatttctact gttgtagata agcttggctg ttttggcgga tgagagaaga ttttcagcct
     1501 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     1561 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     1621 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     1681 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     1741 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     1801 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     1861 cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat
     1921 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1981 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2041 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2101 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2161 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2221 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2281 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2341 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2401 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2461 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2521 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2581 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2641 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2701 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2761 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2821 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2881 gatttacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2941 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3001 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3061 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     3121 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3181 tagtggactc ttgttccaaa cttgaacaac actcaaccct atctcgggct attcttttga
     3241 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3301 atttaacgcg aattttaaca aaatattaac gtttacaatt taaaaggatc taggtgaaga
     3361 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3421 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3481 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3541 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3601 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3661 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3721 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3781 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3841 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3901 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3961 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4021 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4081 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4141 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4201 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4261 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4321 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4381 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4441 tgtgacg
//
