LOCUS       sequence-452297-        7352 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 7352)
  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 7352)
  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-36kb-USF
FEATURES             Location/Qualifiers
     source          1..7352
                     /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          3487..3714
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3732..4433
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4458..4475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4464..4671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4675..4744
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   4747..4887
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4747..4876
                     /label=AAV2 ITR
     rep_origin      4962..5417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5049..5068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5259..5280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5434..5453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5482..5505)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5879..5898
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5968..6015
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6007..6025
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6029..6038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6035..6838
                     /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(6098..6117)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6708..6727
                     /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 aaacaccgcc catgtatggt
     1021 agatgaacgt 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 acaccgcaga cactgccact gtacctgttt 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 accgctttgc atggaatttc ccttgtttaa 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 cgatctgcac tggcacaagg
     2161 tcgtttaaga 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 agtatctgcc tgacggaccc 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 gggggggggg cgcgcgcsag gsgggggggg gsggggsgag
     3361 gggsggggsg gggsgaggsg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     3421 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     3481 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     3541 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     3601 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
     3661 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
     3721 ccggtgccac catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg
     3781 gcaccgtgga caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     3841 gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca
     3901 tcctggctac tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc
     3961 ccgacttctt caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg
     4021 atgacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     4081 acaacgtcaa gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa
     4141 cactcggctg ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca
     4201 gaaacgacat ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca
     4261 catatagatc caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact
     4321 acagactgga aagaatcaag gaggccaaca acgaaaccta cgtcgagcag cacgaggtgg
     4381 cagtggccag atactgcgac ctccctagca aactggggca caagcttaat taagaattcc
     4441 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4501 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4561 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4621 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agctctcgaa
     4681 gccgcggtgc gggtgccagg gcgtgccctt gggctccccg ggcgcgtact ccacctcacc
     4741 catcgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4801 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4861 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4921 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4981 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     5041 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5101 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5161 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5221 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5281 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5341 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5401 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtg
     5461 cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac
     5521 acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg
     5581 ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc
     5641 gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
     5701 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
     5761 tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc
     5821 gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca
     5881 ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc
     5941 ctttcgaccg atcatcaagc tgcacgtgtt gacaattaat catcggcata gtatatcggc
     6001 atagtataat acgactcact ataggagggc caccatggga tcggccattg aacaagatgg
     6061 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     6121 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     6181 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     6241 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     6301 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     6361 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     6421 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     6481 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     6541 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     6601 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     6661 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     6721 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     6781 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgatc
     6841 gagaaattga tgatctatta aacaataaag acgtccacat ggaagtgcaa actattaact
     6901 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6961 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7021 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     7081 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7141 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7201 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7261 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7321 tcagaccccg tagaaaagat caaaggatct tc
//
