LOCUS       sequence-452295-        7351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  5-mer gRNA array targeting Sp dCas9/Cas9 to the Prdm8-Fgf5 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7351)
  AUTHORS   Jensen CL, Chen LF, Swigut T, Crocker OJ, Yao D, Bassik MC, Ferrell 
            JE Jr, Boettiger AN, Wysocka J
  TITLE     Long-range regulation of transcription scales with genomic distance 
            in a gene-specific manner.
  JOURNAL   Mol Cell. 2025 Jan 16;85(2):347-361.e7. doi: 
            10.1016/j.molcel.2024.10.021. Epub 2024 Dec 2.
  PUBMED    39626660
REFERENCE   2  (bases 1 to 7351)
  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: 
            "10.1016/j.molcel.2024.10.021"; journalName: "Mol Cell"; date: 
            "2025-01-16- 16"; volume: "85"; issue: "2"; pages: "347-361.e7"
COMMENT     Sequence Label: pk335-CARGO-5mer-30kb-DSF
FEATURES             Location/Qualifiers
     source          1..7351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2918..3203
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          3486..3713
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3731..4432
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4457..4474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4463..4670
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4674..4743
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   4746..4886
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4746..4875
                     /label=AAV2 ITR
     rep_origin      4961..5416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5048..5067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5258..5279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5433..5452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5481..5504)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5878..5897
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5967..6014
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6006..6024
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6028..6037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6034..6837
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6097..6116)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6707..6726
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gtttaagagc
      661 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
      721 ggcaccgagt cggtgctttt tttctcgagt actaggatcc attaggcttt cccatgattc
      781 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      841 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      901 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      961 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgag tgcaaatgaa
     1021 cctcccccgt ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg
     1081 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tctcgagtac taggatccat
     1141 taggctttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1201 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1261 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1321 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1381 acaccggatg ctgctgagtc tactatgttt aagagctatg ctggaaacag catagcaagt
     1441 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc
     1501 tcgagtacta ggatccatta ggctttccca tgattccttc atatttgcat atacgataca
     1561 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     1621 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     1681 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1741 tcttgtggaa aggacgaaac accggaaagg ctggcggaaa ccacgtttaa gagctatgct
     1801 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
     1861 gagtcggtgc tttttttctc gagtactagg atccattagg ctttcccatg attccttcat
     1921 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1981 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2041 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2101 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgctctgagc acacccccag
     2161 gggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     2221 acttgaaaaa gtggcaccga gtcggtgctt tttttctcga gtactaggat ccattaggct
     2281 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     2341 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     2401 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     2461 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     2521 ccacagagcc agctacttgc gtttaagagc tatgctggaa acagcatagc aagtttaaat
     2581 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctcgagt
     2641 actaggatcc attaggcttt cccatgattc cttcatattt gcatatacga tacaaggctg
     2701 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2761 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2821 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2881 ggaaaggacg aaacaccgtt ttttttctag aggtacccgt tacataactt acggtaaatg
     2941 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag
     3001 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     3061 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     3121 cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     3181 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     3241 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     3301 cgatgggggc gggggggggg gggggggggc gcgcgssagg sggggggggg gggggsgagg
     3361 ggsggggsgg ggsgaggsgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
     3421 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
     3481 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
     3541 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     3601 ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg
     3661 gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac
     3721 cggtgccacc atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     3781 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     3841 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     3901 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     3961 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     4021 tgacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     4081 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     4141 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     4201 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     4261 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     4321 cagactggaa agaatcaagg aggccaacaa cgaaacctac gtcgagcagc acgaggtggc
     4381 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt aagaattcct
     4441 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4501 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4561 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4621 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gctctcgaag
     4681 ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc
     4741 atcgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4801 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4861 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4921 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4981 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     5041 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5101 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5161 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5221 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5281 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5341 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5401 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtgc
     5461 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     5521 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     5581 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     5641 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     5701 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     5761 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     5821 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     5881 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     5941 tttcgaccga tcatcaagct gcacgtgttg acaattaatc atcggcatag tatatcggca
     6001 tagtataata cgactcacta taggagggcc accatgggat cggccattga acaagatgga
     6061 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     6121 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     6181 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     6241 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     6301 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     6361 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     6421 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     6481 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     6541 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     6601 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     6661 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     6721 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     6781 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgatcg
     6841 agaaattgat gatctattaa acaataaaga cgtccacatg gaagtgcaaa ctattaactg
     6901 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6961 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     7021 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7081 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7141 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7201 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7261 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7321 cagaccccgt agaaaagatc aaaggatctt c
//
