LOCUS       sequence-258106-        4409 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 4409)
  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 4409)
  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..4409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             228..239
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             246..959
                     /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      971..1042
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1058..1085
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1176..1193)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1346..1432
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1524..1551
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1570..1661
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1662..1730,1731..2522)
                     /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(1880..1899)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2564..3019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2651..2670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2861..2882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3130..3718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3619..3638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3872..3889
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3904..4044
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3970..3989)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4130..4152
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4290..4309
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(4404..4409,1..29)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
ORIGIN
        1 gctagctcag tcctaggtat aatgctagcg ctagcgaatt cgagctcggt accaagacgt
       61 caactcgacc ctcgagacgg aaagtgaaac gtgatttcat gcgtcatttt gaacattttg
      121 taaatcttat ttaataatgt gtgcggcaat tcacatttaa tttatgaatg ttttcttaac
      181 atcgcggcaa ctcaagaaac ggcaggttcg gatcttagct actagagaaa gaggagaaat
      241 actagatgcg taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg
      301 atggtgatgt caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta
      361 atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga
      421 ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga
      481 agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt
      541 cctttaagga tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc
      601 tggtaaaccg cattgagctg aaaggcattg actttaaaga ggacggcaat atcctgggcc
      661 ataagctgga atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa
      721 atggcattaa agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg
      781 ctgatcacta ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc
      841 actatctgag cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg
      901 ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat
      961 gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1021 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
     1081 ctgcgtttat acgtctctaa cggtcaaaag acctttttaa tttctactct tgtagataag
     1141 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
     1201 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     1261 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     1321 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1381 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     1441 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     1501 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     1561 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1621 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1681 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1741 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1801 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1861 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1921 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1981 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2041 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2101 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2161 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2221 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2281 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2341 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2401 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2461 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2521 aactgtcaga ccaagtttac tcatatatac tttagattga tttacgcgcc ctgtagcggc
     2581 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2641 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2701 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2761 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2821 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2881 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     2941 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3001 atattaacgt ttacaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3061 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3121 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3181 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3241 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3301 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3361 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3421 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3481 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3541 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3601 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3661 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3721 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3781 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3841 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3901 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     3961 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4021 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4081 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgacggtctc cgggagctgc
     4141 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagaagga gatggcgccc
     4201 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     4261 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     4321 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat ctgctcatgt
     4381 ttgacagctt atcatcgatg catttgaca
//
