LOCUS       sequence-271456-        4723 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expression of Casilio sgRNAs containing five 
            PUFa-binding sites (PBSa).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4723)
  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 4723)
  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: pAT1503_pX-SPFE-BbsI(ccdbCam)-5xPBSa
FEATURES             Location/Qualifiers
     source          1..4723
                     /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"
     CDS             complement(306..611)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(408..427)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(953..1612)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1527..1546
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1666..1672,1673..1690,1691..1696))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     repeat_region   1986..2126
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1986..2115
                     /label=AAV2 ITR
     rep_origin      2201..2656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2288..2307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2498..2519
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2673..2692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2792..2814
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2852..2870)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2938..3042
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3043..3111,3112..3903)
                     /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(3261..3280)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4074..4662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4563..4582
                     /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 tcttcgtcga cctgcagact ggctgtgtat aagggagcct
      301 gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg
      361 ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc
      421 atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta
      481 tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata
      541 atatcactct gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc
      601 ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca tttcaataaa
      661 ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg cacgcagacg
      721 acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat catatatgcc
      781 ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt catagcatac
      841 ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg caaaaatcag
      901 cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggatccagat ctttacgccc
      961 cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc
     1021 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     1081 tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta
     1141 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa
     1201 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt
     1261 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt
     1321 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt
     1381 tcattgccat acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg
     1441 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct
     1501 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac
     1561 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt
     1621 ccttagctcc tgaaaatctc gacggatcct aactcaaaat ccacacatta tacgagccgg
     1681 aagcataaag tgtaaagcct ggggtgccta atgcggccgc gaagacctgt ttaagagcta
     1741 tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg
     1801 caccgagtcg gtgccaattg ggtctccaga ttgtatgtag cctgtatgta gcctgtatgt
     1861 agcctgtatg tagcctgtat gtaagatctt tttttgtttt agagctagaa atagcaagtt
     1921 aaaataaggc tagtccgtag cgcgtgcgcc aattctgcag acaaatggcg gcgcgccgcg
     1981 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     2041 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     2101 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     2161 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     2221 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     2281 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2341 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2401 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2461 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2521 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     2581 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2641 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2701 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2761 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2821 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     2881 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     2941 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3001 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3061 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3121 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3181 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3241 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3301 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3361 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3421 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3481 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3541 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3601 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3661 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3721 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     3781 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3841 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3901 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3961 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4021 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4081 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4141 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4201 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4261 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4321 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4381 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4441 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4501 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4561 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4621 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4681 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
