LOCUS       sequence-452298-        6972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4-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 6972)
  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 6972)
  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-4mer-Fgf5Pro
FEATURES             Location/Qualifiers
     source          1..6972
                     /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        2540..2825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2827..3106
                     /label=chicken beta-actin promoter
     intron          3107..3334
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3352..4053
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4078..4095)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4084..4291
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4295..4364
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   4367..4507
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4367..4496
                     /label=AAV2 ITR
     rep_origin      4582..5037
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4669..4688)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4879..4900
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5054..5073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5102..5125)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5499..5518
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5588..5635
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5627..5645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5649..5658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5655..6458
                     /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(5718..5737)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6328..6347
                     /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 tcgcagccag
     1021 cgtcccgggt 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 cgatcccggg agtggggttt 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 accgcacccc cgcccccggg accggtttaa 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 cgccgctttg tgtactcacg
     2161 gcgtttaaga 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 ttttttttct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2581 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
     2641 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2701 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
     2761 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2821 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     2881 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
     2941 gggggggggg cgcgcgcsag gsggggsggg gsggggsgag gggsggggsg gggsgaggcg
     3001 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     3061 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     3121 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     3181 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     3241 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     3301 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgagcgag
     3361 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     3421 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     3481 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     3541 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     3601 ttccctgagg gcttcacatg ggagagagtc accacatacg atgacggggg cgtgctgacc
     3661 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     3721 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     3781 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     3841 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc
     3901 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     3961 gaggccaaca acgaaaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     4021 ctccctagca aactggggca caagcttaat taagaattcc tagagctcgc tgatcagcct
     4081 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4141 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4201 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4261 attgggaaga gaatagcagg catgctgggg agctctcgaa gccgcggtgc gggtgccagg
     4321 gcgtgccctt gggctccccg ggcgcgtact ccacctcacc catcgcagga acccctagtg
     4381 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4441 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4501 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4561 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4621 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4681 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4741 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4801 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4861 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     4921 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     4981 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5041 ggtgcactct cagtacaatc tgctctgatg ccgcatagtg cttttcccaa ggcagtctgg
     5101 agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg
     5161 cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc
     5221 gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
     5281 caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc
     5341 tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc
     5401 tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
     5461 gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg
     5521 cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgaccg atcatcaagc
     5581 tgcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgactcact
     5641 ataggagggc caccatggga tcggccattg aacaagatgg attgcacgca ggttctccgg
     5701 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     5761 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     5821 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     5881 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     5941 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     6001 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     6061 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     6121 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     6181 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     6241 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     6301 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     6361 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     6421 gcatcgcctt ctatcgcctt cttgacgagt tcttctgatc gagaaattga tgatctatta
     6481 aacaataaag acgtccacat ggaagtgcaa actattaact ggcgaactac ttactctagc
     6541 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6601 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     6661 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6721 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6781 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6841 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6901 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6961 caaaggatct tc
//
