LOCUS       sequence-450827-        5239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BACH1 targeting crRNA array for RfxCas13d expressed from multiple 
            hU6 promoter and reporter BFP protein expressed from EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5239)
  AUTHORS   Wan Y, Helenek C, Coraci D, Balazsi G
  TITLE     Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA 
            Downregulation with Minimal Collateral RNA Cutting.
  JOURNAL   ACS Synth Biol. 2024 Oct 18;13(10):3212-3230. doi: 
            10.1021/acssynbio.4c00271. Epub 2024 Oct 8.
  PUBMED    39377757
REFERENCE   2  (bases 1 to 5239)
  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"; doi: 
            "10.1021/acssynbio.4c00271"; journalName: "ACS Synth Biol"; date: 
            "2024-10-18- 18"; volume: "13"; issue: "10"; pages: "3212-3230"
COMMENT     Sequence Label: pUC-hU6-crBACH1-array_EF1a-BFP
FEATURES             Location/Qualifiers
     source          1..5239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    96..320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     309..326
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        complement(1069..2247)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1078..2016
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1101..1121)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_RNA        complement(2302..2337)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        complement(2361..2396)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        complement(2420..2455)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        complement(2457..2705)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        complement(2769..2804)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        complement(2828..2863)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        complement(2865..3113)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        complement(3177..3212)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        complement(3236..3271)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        complement(3273..3521)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      complement(3584..4172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3664..3683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4343..5134,5135..5203))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4966..4985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5204..5239,1..69))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
       61 ggttccgcgc acatttcccc taccctaaat ctagaccata gagcccaccg catccccagc
      121 atgcctgcta ttatcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
      181 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
      241 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
      301 ctggcaacta gaaggcacag tcgaggctga ttagcgagct ctagctgatc aattcagctt
      361 gtgccccaat ttgctcggca gatcgcagta gcgtgcaact gcaacctcat gttgctccac
      421 ataggtttcg ttgttagctt cttttattct ctcaagccta tagtcgacgt aatatacacc
      481 aggcatcttc aaatttttgg cgggcttttt agaccgatag gtcgtcttta tattcgctat
      541 aaggtggctc ccgccaacca atttcaatgc catgtcgttt cggccttcca aaccaccgtc
      601 ggctgggtaa agagtctctg taaaagcctc ccagccgagg gtttttttct gcataactgg
      661 gccgtttgaa gtaaagttta cgcctcgaat cttcacattg tagatcaaac acccgtcttg
      721 caggctggtg tcctgagtag cggtcagcac gccgccgtcc tcgtatgttg tgacccgctc
      781 ccaagtaaac ccctcgggaa atgactgctt aaagaagtct ggaatgcctt gcgtatggtt
      841 gataaaagtt ttactcccgt agagaaaaga agttgccaga atgtcaaagg caaagggcag
      901 cggcccacct tctactactt taattctcat cgtttgggtg ccctcgtagg gtttgccctc
      961 tccttcggag gtacatttga aatggtggtt gtcgaccgta ccttccatat aaagtttcat
     1021 gtgcatgttc tctttaatca gttcggacat gctcatggtg gccgtacgtc acgacacctg
     1081 aaatggaaga aaaaaacttt gaaccactgt ctgaggcttg agaatgaacc aagatccaaa
     1141 ctcaaaaagg gcaaattcca aggagaatta catcaagtgc caagctggcc taacttcagt
     1201 ctccacccac tcagtgtggg gaaactccat cgcataaaac ccctcccccc aacctaaaga
     1261 cgacgtactc caaaagctcg agaactaatc gaggtgcctg gacggcgccc ggtactccgt
     1321 ggagtcacat gaagcgacgg ctgaggacgg aaaggccctt ttcctttgtg tgggtgactc
     1381 acccgcccgc tctcccgagc gccgcgtcct tcattttgag ctccctgcag cagggccggg
     1441 aagcggccat ctttccgctc acgcaactgg tgccgaccgg gccagccttg ccgcccaggg
     1501 cggggcgata cacggcggcg cgaggccagg caccagagca ggccggccag cttgagacta
     1561 cccccgtccg attctcggtg gccgcgctcg caggccccgc ctcgccgaac atgtgcgctg
     1621 ggacgcacgg gccccgtcgc cgcccgcggc cccaaaaacc gaaataccag tgtgcagatc
     1681 ttggcccgca tttacaagac tatcttgcca gaaaaaaagc gtcgcagcag gtcatcaaaa
     1741 attttaaatg gctagagact tatcgaaagc agcgagacag gcgcgaaggt gccaccagat
     1801 tcgcacgcgg cggccccagc gcccaggcca ggcctcaact caagcacgag gcgaaggggc
     1861 tccttaagcg caaggcctcg aactctccca cccacttcca acccgaagct cgggatcaag
     1921 aatcacgtac tgcagccagg tggaagtaat tcaaggcacg caagggccat aacccgtaaa
     1981 gaggccaggc ccgcgggaac cacacacggc acttacctgt gttctggcgg caaacccgtt
     2041 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     2101 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     2161 ggcaccggtt caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg
     2221 ctctgcccac tgacgggcac cggagccgaa ttcccactgg ggagttctag tacttgatac
     2281 gatacatagg tgaaaaaaaa agtttcaaac cccgaccagt tggtaggggt ttacttggtg
     2341 gtgggatctc agatttctgt gtttcaaacc ccgaccagtt ggtaggggtt tacttggtgg
     2401 tgggatctca gatttctgtg tttcaaaccc cgaccagttg gtaggggttt acttgcggtg
     2461 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     2521 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     2581 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcca
     2641 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     2701 ccctcatgaa catggttagc agagggggga gttctagtac ttgatacgat acataggtga
     2761 aaaaaaaagt ttcaaacccc gaccagttgg taggggttta cttgctgtct cgaaatgatt
     2821 ttcagtcgtt tcaaaccccg accagttggt aggggtttac ttgcggtgtt tcgtcctttc
     2881 cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag
     2941 tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt
     3001 cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta
     3061 acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctccaccctg
     3121 aaaactttgc cccggggagt tctagtactt gatacgatac ataggtgaaa aaaaaagttt
     3181 caaaccccga ccagttggta ggggtttact tgctctggcc ttgccttctt tatgcgtttc
     3241 aaaccccgac cagttggtag gggtttactt gcggtgtttc gtcctttcca caagatatat
     3301 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     3361 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     3421 actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat
     3481 cgtatatgca aatatgaagg aatcatggga aataggccct cgctagctga gcaaaaggcc
     3541 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3601 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3661 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3721 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3781 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3841 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3901 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3961 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4021 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4081 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4141 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4201 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4261 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4321 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4381 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4441 gggagggctt accatctggc cccagtgctg caatgatacc gcggcttcca cgctcaccgg
     4501 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4561 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4621 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4681 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4741 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4801 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4861 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4921 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4981 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5041 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5101 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5161 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5221 attattgaag catttatca
//
