LOCUS       sequence-445692-        4379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA scaffold for RfxCas13d expressed from hU6-2xTetO 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 4379)
  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 4379)
  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-hU6-2O-crScaffold_EF1a-BFP
FEATURES             Location/Qualifiers
     source          1..4379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      81..669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     570..589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     732..752
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     903..922
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    933..951
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1002..1037
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        1056..1091
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        1146..2324
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1377..2315
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2272..2292
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(3067..3084)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3073..3297
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3324..3428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3429..3497,3498..4289)
                     /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     complement(3647..3666)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
       61 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
      121 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
      181 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
      241 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
      301 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
      361 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
      421 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
      481 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
      541 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
      601 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
      661 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
      721 gctcagctag cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      781 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      841 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      901 tggactatca tatgcttacc gtaacttgaa actccctatc agtgatagag attatatatc
      961 tccctatcag tgatagacac cgtggaaagg acgaaacacc gcaagtaaac ccctaccaac
     1021 tggtcggggt ttgaaacggg tcttcgagaa gacctcaagt aaacccctac caactggtcg
     1081 gggtttgaaa cttttttttt cacctatgta tcgtatcaag tactagaact ccccagtggg
     1141 aattcggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     1201 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     1261 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     1321 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     1381 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     1441 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
     1501 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
     1561 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
     1621 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
     1681 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
     1741 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
     1801 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
     1861 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
     1921 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     1981 gaaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     2041 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     2101 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     2161 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     2221 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     2281 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgacgtacg gccaccatga
     2341 gcatgtccga actgattaaa gagaacatgc acatgaaact ttatatggaa ggtacggtcg
     2401 acaaccacca tttcaaatgt acctccgaag gagagggcaa accctacgag ggcacccaaa
     2461 cgatgagaat taaagtagta gaaggtgggc cgctgccctt tgcctttgac attctggcaa
     2521 cttcttttct ctacgggagt aaaactttta tcaaccatac gcaaggcatt ccagacttct
     2581 ttaagcagtc atttcccgag gggtttactt gggagcgggt cacaacatac gaggacggcg
     2641 gcgtgctgac cgctactcag gacaccagcc tgcaagacgg gtgtttgatc tacaatgtga
     2701 agattcgagg cgtaaacttt acttcaaacg gcccagttat gcagaaaaaa accctcggct
     2761 gggaggcttt tacagagact ctttacccag ccgacggtgg tttggaaggc cgaaacgaca
     2821 tggcattgaa attggttggc gggagccacc ttatagcgaa tataaagacg acctatcggt
     2881 ctaaaaagcc cgccaaaaat ttgaagatgc ctggtgtata ttacgtcgac tataggcttg
     2941 agagaataaa agaagctaac aacgaaacct atgtggagca acatgaggtt gcagttgcac
     3001 gctactgcga tctgccgagc aaattggggc acaagctgaa ttgatcagct agagctcgct
     3061 aatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3121 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3181 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3241 agggggagga ttgggaagat aatagcaggc atgctgggga tgcggtgggc tctatggtct
     3301 agatttaggg taggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3361 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3421 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3481 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3541 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3601 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3661 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3721 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3781 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3841 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3901 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3961 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4021 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4081 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4141 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4201 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4261 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4321 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgat
//
