LOCUS       sequence-452270-        7732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  6-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 7732)
  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 7732)
  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-6mer-26kb-DSF
FEATURES             Location/Qualifiers
     source          1..7732
                     /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        3296..3581
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          3867..4094
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4112..4813
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4838..4855)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4844..5051
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5055..5124
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   5127..5267
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5127..5256
                     /label=AAV2 ITR
     rep_origin      5342..5797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5429..5448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5639..5660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5814..5833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5862..5885)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6259..6278
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        6348..6395
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6387..6405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6409..6418
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6415..7218
                     /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(6478..6497)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7088..7107
                     /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 aaacaccggg aagatttgca
     1021 gatcataagt 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 acaccgctcc atccactgtt tgccatgttt 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 accgtgtctg agtcagttgg tcgggtttaa 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 cggtgtcagg gggtctcaag
     2161 ctgtttaaga 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 ttagccagtt gttaagtctc 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 aaacaccgaa tggttggagc agcatcatgt ttaagagcta tgctggaaac
     2941 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3001 gtgctttttt tctcgagtac taggatccat taggctttcc catgattcct tcatatttgc
     3061 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3121 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3181 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3241 tggctttata tatcttgtgg aaaggacgaa acaccgtttt ttttctagag gtacccgtta
     3301 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3361 caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     3421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     3481 atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg gactttccta
     3541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
     3601 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
     3661 tttaattatt ttgtgcagcg atgggggcgg gggggggggg gggggggggs cgsgsgssag
     3721 gggggggggg gsggggsgag gggsggggsg gggsgaggsg gagaggtgcg gcggcagcca
     3781 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     3841 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
     3901 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
     3961 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
     4021 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
     4081 tcactttttt tcaggttgga ccggtgccac catgagcgag ctgattaagg agaacatgca
     4141 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     4201 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     4261 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     4321 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     4381 ggagagagtc accacatacg atgacggggg cgtgctgacc gctacccagg acaccagcct
     4441 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     4501 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     4561 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     4621 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     4681 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgaaaccta
     4741 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     4801 caagcttaat taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     4861 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4921 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4981 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     5041 catgctgggg agctctcgaa gccgcggtgc gggtgccagg gcgtgccctt gggctccccg
     5101 ggcgcgtact ccacctcacc catcgcagga acccctagtg atggagttgg ccactccctc
     5161 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     5221 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     5281 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     5341 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5401 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5461 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5521 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     5581 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5641 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     5701 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     5761 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5821 tgctctgatg ccgcatagtg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     5881 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     5941 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     6001 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     6061 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     6121 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     6181 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     6241 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     6301 cctcttcctc atctccgggc ctttcgaccg atcatcaagc tgcacgtgtt gacaattaat
     6361 catcggcata gtatatcggc atagtataat acgactcact ataggagggc caccatggga
     6421 tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     6481 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     6541 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     6601 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     6661 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     6721 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     6781 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     6841 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     6901 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     6961 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     7021 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     7081 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     7141 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     7201 cttgacgagt tcttctgatc gagaaattga tgatctatta aacaataaag acgtccacat
     7261 ggaagtgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     7321 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     7381 ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac
     7441 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     7501 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     7561 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     7621 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7681 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
