LOCUS       sequence-271477-        3344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expression of sgRNAs with dual PUF-Binding Sites 
            (5xPBSa - 5xPBSc) of Casilio-ME 2.3, 2.4, 3.3 and 3.4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3344)
  AUTHORS   Taghbalout A, Du M, Jillette N, Rosikiewicz W, Rath A, Heinen CD, Li
            S, Cheng AW
  TITLE     Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated 
            RNA-guided coupling of methylcytosine oxidation and DNA repair 
            pathways.
  JOURNAL   Nat Commun. 2019 Sep 20;10(1):4296. doi: 10.1038/s41467-019-12339-7.
  PUBMED    31541098
REFERENCE   2  (bases 1 to 3344)
  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"; journalName: "Nat 
            Commun."; date: "2019-09-20"; volume: "10(1):4296. doi"; pages: " 
            10.1038/s41467-019-12339-7"
COMMENT     Sequence Label: pAT243_pX-sgRNA-5xPBSa-5xPBSc
FEATURES             Location/Qualifiers
     source          1..3344
                     /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   607..747
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   607..736
                     /label=AAV2 ITR
     rep_origin      822..1277
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(909..928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1119..1140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1294..1313)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1413..1435
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1473..1491)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1559..1663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1664..1732,1733..2524)
                     /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(1882..1901)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2695..3283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3184..3203
                     /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 ctccagattg tatgtagcct gtatgtagcc tgtatgtagc ctgtatgtag
      421 cctgtatgta agatccaatt gggtctccag atttgatgta gccttgatgt agccttgatg
      481 tagccttgat gtagccttga tgtaagatct ttttttgttt tagagctaga aatagcaagt
      541 taaaataagg ctagtccgta gcgcgtgcgc caattctgca gacaaatggc ggcgcgccgc
      601 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
      661 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
      721 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
      781 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
      841 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
      901 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      961 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1021 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1081 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1141 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     1201 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1261 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     1321 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1381 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1441 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     1501 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1561 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1621 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1681 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1741 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1801 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1861 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1921 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1981 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2041 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2101 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2161 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     2221 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2281 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2341 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     2401 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2461 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2521 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2581 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2641 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2701 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2761 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2821 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2881 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2941 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3001 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3061 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3121 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3181 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3241 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3301 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
