LOCUS       sequence-445927-        4831 bp    DNA     circular SYN 17-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 4831)
  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 4831)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 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..4831
                     /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)"
     rep_origin      complement(3176..3764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3256..3275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3935..4726,4727..4795))
                     /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     4558..4577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4796..4831,1..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 ctcgctagct
     3121 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3181 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3241 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3301 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3361 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3421 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3481 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3541 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3601 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3661 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3721 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3781 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3841 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3901 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3961 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4021 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcggcttc
     4081 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4141 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4201 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4261 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4321 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4381 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4441 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4501 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4561 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4621 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4681 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4741 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4801 tcctttttca atattattga agcatttatc a
//
