LOCUS       sequence-452312-        6594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3-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 6594)
  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 6594)
  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-3mer-36kb-USF
FEATURES             Location/Qualifiers
     source          1..6594
                     /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        2162..2447
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2449..2728
                     /label=chicken beta-actin promoter
     intron          2729..2956
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2974..3675
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(3700..3717)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3706..3913
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3917..3986
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   3989..4129
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3989..4118
                     /label=AAV2 ITR
     rep_origin      4204..4659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4291..4310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4501..4522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4676..4695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4724..4747)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5121..5140
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5210..5257
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5249..5267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5271..5280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5277..6080
                     /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(5340..5359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5950..5969
                     /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 aaacaccgct ttgcatggaa
     1021 tttcccttgt 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 acaccgatct gcactggcac aaggtcgttt 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 accgagtatc tgcctgacgg acccgtttaa 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 cgtttttttt ctagaggtac
     2161 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2221 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2281 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2341 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
     2401 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     2461 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     2521 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg ggcgcgcgcs
     2581 aggsggggsg gggsggggsg aggggsgggg sggggsgagg sggagaggtg cggcggcagc
     2641 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
     2701 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
     2761 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     2821 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
     2881 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     2941 aatcactttt tttcaggttg gaccggtgcc accatgagcg agctgattaa ggagaacatg
     3001 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     3061 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     3121 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     3181 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     3241 tgggagagag tcaccacata cgatgacggg ggcgtgctga ccgctaccca ggacaccagc
     3301 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     3361 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
     3421 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     3481 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     3541 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgaaacc
     3601 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     3661 cacaagctta attaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     3721 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3781 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3841 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     3901 ggcatgctgg ggagctctcg aagccgcggt gcgggtgcca gggcgtgccc ttgggctccc
     3961 cgggcgcgta ctccacctca cccatcgcag gaacccctag tgatggagtt ggccactccc
     4021 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4081 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4141 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4201 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4261 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4381 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4501 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     4561 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     4621 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4681 tctgctctga tgccgcatag tgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     4741 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     4801 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     4861 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     4921 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     4981 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     5041 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
     5101 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     5161 ctcctcttcc tcatctccgg gcctttcgac cgatcatcaa gctgcacgtg ttgacaatta
     5221 atcatcggca tagtatatcg gcatagtata atacgactca ctataggagg gccaccatgg
     5281 gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5341 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     5401 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     5461 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     5521 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     5581 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5641 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5701 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5761 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     5821 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5881 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5941 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6001 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6061 ttcttgacga gttcttctga tcgagaaatt gatgatctat taaacaataa agacgtccac
     6121 atggaagtgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6241 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     6301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
