LOCUS       sequence-134409-        3494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expression of sgRNA-25xPBSb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3494)
  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 3494)
  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: pAC1376-pX-sgRNA-25xPBSb
FEATURES             Location/Qualifiers
     source          1..3494
                     /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   757..897
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   757..886
                     /label=AAV2 ITR
     rep_origin      972..1427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1059..1078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1269..1290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1444..1463)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1563..1585
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1623..1641)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1709..1813
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1814..1882,1883..2674)
                     /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(2032..2051)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2845..3433
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3334..3353
                     /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 agatttgata tagccttgat atagccttga tatagccttg atatagcctt
      481 gatataagat ttgatatagc cttgatatag ccttgatata gccttgatat agccttgata
      541 taagatttga tatagccttg atatagcctt gatatagcct tgatatagcc ttgatataag
      601 atttgatata gccttgatat agccttgata tagccttgat atagccttga tataagatct
      661 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgta gcgcgtgcgc
      721 caattctgca gacaaatggc ggcgcgccgc ggccgcagga acccctagtg atggagttgg
      781 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
      841 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
      901 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
      961 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1021 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1081 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1141 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     1201 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1261 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
     1321 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1381 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     1441 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     1501 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     1561 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     1621 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1681 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1741 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1801 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1861 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1921 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1981 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2041 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2101 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2161 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2221 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2281 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2341 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2401 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2461 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2521 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2581 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2641 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2701 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2761 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2821 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2881 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2941 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3001 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3061 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3121 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3181 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3241 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3301 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3361 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3421 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3481 ttttgctcac atgt
//
