LOCUS       sequence-134403-        3214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expression of sgRNA without PBS. It contains 
            extra sequences for BsaI digestion for insertion of PBS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3214)
  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 3214)
  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: pAC1394-pX-sgRNA-0xPBS
FEATURES             Location/Qualifiers
     source          1..3214
                     /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   477..617
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   477..606
                     /label=AAV2 ITR
     rep_origin      692..1147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(779..798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     989..1010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1164..1183)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1283..1305
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1343..1361)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1429..1533
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1534..1602,1603..2394)
                     /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(1752..1771)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2565..3153
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3054..3073
                     /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 ctccagatct ttttttgttt tagagctaga aatagcaagt taaaataagg
      421 ctagtccgta gcgcgtgcgc caattctgca gacaaatggc ggcgcgccgc ggccgcagga
      481 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
      541 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
      601 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      661 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
      721 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      781 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      841 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      901 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      961 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1021 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1081 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     1141 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1201 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1261 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1321 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1381 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1441 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1501 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1561 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1621 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1681 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1741 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1801 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1861 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1921 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     1981 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2041 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2101 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2161 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2221 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     2281 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2341 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2401 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2461 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2521 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2581 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2641 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2701 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2761 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2821 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2881 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2941 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3001 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3061 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3121 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3181 ttcctggcct tttgctggcc ttttgctcac atgt
//
