LOCUS       sequence-134364-        3438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expression of sgRNA-20xPBSb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3438)
  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 3438)
  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: pAC1425-pX-sgRNA-20xPBSb
FEATURES             Location/Qualifiers
     source          1..3438
                     /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   701..841
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   701..830
                     /label=AAV2 ITR
     rep_origin      916..1371
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1003..1022)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1213..1234
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1388..1407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1507..1529
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1567..1585)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1653..1757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1758..1826,1827..2618)
                     /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(1976..1995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2789..3377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3278..3297
                     /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 atcttttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtagcgcgt
      661 gcgccaattc tgcagacaaa tggcggcgcg ccgcggccgc aggaacccct agtgatggag
      721 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
      781 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
      841 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
      901 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      961 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1021 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1081 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     1141 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1201 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     1261 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1321 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     1381 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     1441 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     1501 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     1561 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1621 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1681 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1741 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1801 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1861 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1921 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1981 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2041 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2101 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2161 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2221 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2281 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2341 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2401 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2461 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2521 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2581 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2641 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2701 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2761 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2821 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2881 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2941 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3001 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3061 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3121 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3181 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3241 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3301 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3361 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3421 ggccttttgc tcacatgt
//
