LOCUS       sequence-252348-        4410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for generating CRISPR arrays for AsCas12a. 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: pAsCpf1GG
FEATURES             Location/Qualifiers
     source          1..4410
                     /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)"
     RBS             513..524
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             531..1244
                     /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      1256..1327
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1343..1370
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1461..1478)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1631..1717
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1809..1836
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1855..1946
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1947..2015,2016..2807)
                     /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(2165..2184)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2849..3304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2936..2955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3415..4003
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3904..3923
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4157..4174
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4189..4329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4255..4274)
                     /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 cgaccctcga
      361 gacggaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat cttatttaat
      421 aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc ggcaactcaa
      481 gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag atgcgtaaag
      541 gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg
      601 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
      661 tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc
      721 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
      781 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
      841 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
      901 agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca
      961 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
     1021 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
     1081 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
     1141 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
     1201 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat
     1261 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1321 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatacgtc
     1381 tctaacggtc aaaagacctt tttaatttct actcttgtag ataagcttgg ctgttttggc
     1441 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata
     1501 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     1561 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     1621 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1681 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt
     1741 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca
     1801 aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt
     1861 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1921 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1981 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2041 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2101 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2161 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     2221 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2281 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2341 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2401 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2461 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     2521 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2581 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2641 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2701 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2761 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2821 tttactcata tatactttag attgatttac gcgccctgta gcggcgcatt aagcgcggcg
     2881 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2941 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3001 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3061 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3121 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaacttgaac aacactcaac
     3181 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3241 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3301 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3361 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3421 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3481 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3541 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3601 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3661 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3721 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3781 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3841 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3901 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3961 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4021 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4081 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4141 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4201 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     4261 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4321 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4381 ccggcatccg cttacagaca agctgtgacg
//
