LOCUS       sequence-257701-        4410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for generating CRISPR arrays for FnCas12a. Contains
            a GFP-dropout cassette and a direct repeat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4410)
  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 4410)
  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: pFnCpf1GG
FEATURES             Location/Qualifiers
     source          1..4410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..52
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             251..262
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             269..982
                     /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      994..1065
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1081..1108
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1200..1217)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1370..1456
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1548..1575
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1594..1685
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1686..1754,1755..2546)
                     /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(1904..1923)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2588..3043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2675..2694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2885..2906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3154..3742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3643..3662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3896..3913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3928..4068
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3994..4013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4154..4176
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4314..4333
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttatcatc gatgcatttg acagctagct cagtcctagg tataatgcta gcgctagcga
       61 attcgagctc ggtaccaaga cgtcaactcg accctcgaga cggaaagtga aacgtgattt
      121 catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc aattcacatt
      181 taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt tcggatctta
      241 gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagctgt tcactggtgt
      301 cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt ccgtgcgtgg
      361 cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct gtactactgg
      421 taaactgccg gttccttggc cgactctggt aacgacgctg acttatggtg ttcagtgctt
      481 tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca tgccggaagg
      541 ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa cgcgtgcgga
      601 agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca ttgactttaa
      661 agaggacggc aatatcctgg gccataagct ggaatacaat tttaacagcc acaatgttta
      721 catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc gccacaacgt
      781 ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa tcggtgatgg
      841 tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt ctaaagatcc
      901 gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg gcatcacgca
      961 tggtatggat gaactgtaca aatgaccagg catcaaataa aacgaaaggc tcagtcgaaa
     1021 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     1081 ggctcacctt cgggtgggcc tttctgcgtt tatacgtctc taacggtcta agaactttaa
     1141 ataatttcta ctgttgtaga taagcttggc tgttttggcg gatgagagaa gattttcagc
     1201 ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc
     1261 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     1321 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     1381 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     1441 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     1501 gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct
     1561 gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa
     1621 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1681 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1741 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1801 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1861 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1921 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     1981 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2041 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2101 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2161 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2221 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2281 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2341 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2401 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2461 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2521 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2581 ttgatttacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2641 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2701 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2761 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     2821 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2881 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     2941 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3001 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaaagga tctaggtgaa
     3061 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3121 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3181 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3241 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3301 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3361 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3421 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3481 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3541 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3601 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3661 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3721 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3781 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3841 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3901 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3961 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4021 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4081 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4141 gctgtgacgg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4201 gcgaggcaga aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     4261 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     4321 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     4381 ccggcgtaga ggatctgctc atgtttgaca
//
