LOCUS       sequence-134411-        3270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for transient expression of sgRNA-5xPBSb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3270)
  AUTHORS   Cheng AW, Jillette N, Lee P, Plaskon D, Fujiwara Y, Wang W, 
            Taghbalout A, Wang H
  TITLE     Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation 
            and genomic labeling.
  JOURNAL   Cell Res. 2016 Feb;26(2):254-7. doi: 10.1038/cr.2016.3. Epub 2016 
            Jan 15.
  PUBMED    26768771
REFERENCE   2  (bases 1 to 3270)
  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: "10.1038/cr.2016.3";
            journalName: "Cell Res"; date: "2016-02"; volume: "26"; issue: "2"; 
            pages: "254-7"
COMMENT     Sequence Label: pAC1374-pX-sgRNA-5xPBSb
FEATURES             Location/Qualifiers
     source          1..3270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..257
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9..29
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     180..199
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   533..673
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   533..662
                     /label=AAV2 ITR
     rep_origin      748..1203
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(835..854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1045..1066
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1220..1239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1339..1361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1399..1417)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1485..1589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1590..1658,1659..2450)
                     /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(1808..1827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2621..3209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3110..3129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcgcgccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
       61 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      121 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      181 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
      241 ggaaaggacg aaacaccggg tcttcgagaa gacctgttta agagctatgc tggaaacagc
      301 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      361 ccaattgggt ctccagattt gatatagcct tgatatagcc ttgatatagc cttgatatag
      421 ccttgatata agatcttttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
      481 tccgtagcgc gtgcgccaat tctgcagaca aatggcggcg cgccgcggcc gcaggaaccc
      541 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
      601 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
      661 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      721 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
      781 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      841 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      901 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      961 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1021 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1081 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1141 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1201 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     1261 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1321 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     1381 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     1441 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1561 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1621 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1681 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1741 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1801 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1861 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1921 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1981 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2041 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2101 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2161 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2221 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2281 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     2341 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2401 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2461 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2521 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2761 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3241 tggccttttg ctggcctttt gctcacatgt
//
