LOCUS       sequence-419895-        4657 bp    DNA     circular SYN 15-APR-2025
DEFINITION  backbone for gRNA cloning of dpspCas13b tagged by 5xPBSc_Loop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4657)
  AUTHORS   Liu Z, Jillette N, Robson P, Cheng AW
  TITLE     Simultaneous multifunctional transcriptome engineering by CRISPR RNA
            scaffold.
  JOURNAL   Nucleic Acids Res. 2023 Aug 11;51(14):e77. doi: 10.1093/nar/gkad547.
  PUBMED    37395412
REFERENCE   2  (bases 1 to 4657)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2023-08-11"; volume: "51(14):e77. doi"; pages: "
            10.1093/nar/gkad547"
COMMENT     Sequence Label: gzk326-dpspCas13b_gRNA[ccdbCam]-5xPBSc_Loop
FEATURES             Location/Qualifiers
     source          1..4657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        683..931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     683..703
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     854..873
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(980..1285)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1082..1101)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1627..2286)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     NNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2201..2220
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2340..2346,2347..2364,2365..2370))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2536..2635)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2652..2671)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2653..2671)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2676..2692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3123..3914
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4007..4595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4496..4515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgatca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtattcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgagggcct atttcccatg attccttcat atttgcatat
      721 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      781 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      841 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      901 tttatatatc ttgtggaaag gacgaaacac cgggtcttcg tcgacctgca gactggctgt
      961 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
     1021 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggacacc ggcacactgg
     1081 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
     1141 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
     1201 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
     1261 tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg
     1321 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
     1381 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
     1441 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
     1501 gcttcatagc atacctcttt tttacatact tcgggtatac atatcagtat atattcttat
     1561 accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc
     1621 agatctttac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     1681 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     1741 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgttcat
     1801 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     1861 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     1921 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     1981 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     2041 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
     2101 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     2161 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     2221 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     2281 ctccatttta gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac
     2341 attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgcgaagac
     2401 ctgttgtgga aggtccagtt ttgaggggct attacaacca attgggtctc cagatttgat
     2461 gtaagggccc attgatgtaa gggcccattg atgtaagatc tttttttgtt ttggcgcgcc
     2521 ttaattaaga attcgaccca gctttcttgt acaaagttgg cattataaaa aataattgct
     2581 catcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc
     2641 cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct
     2701 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgcc
     2761 tcctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt
     2821 gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc
     2881 gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac
     2941 gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg
     3001 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     3061 tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca
     3121 ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca
     3181 tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
     3241 gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg
     3301 aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga
     3361 gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc
     3421 gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag
     3481 gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag
     3541 aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac
     3601 aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
     3661 ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg
     3721 gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt
     3781 atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg
     3841 cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg
     3901 tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt
     3961 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4021 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4081 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4141 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4201 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4261 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4321 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4381 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4441 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4501 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4561 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4621 ggttcctggc cttttgctgg ccttttgctc acatgtt
//
