LOCUS       sequence-452304-        6596 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 6596)
  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 6596)
  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-35kb-DSF
FEATURES             Location/Qualifiers
     source          1..6596
                     /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        2161..2446
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2731..2958
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2976..3677
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(3702..3719)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3708..3915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3919..3988
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   3991..4131
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3991..4120
                     /label=AAV2 ITR
     rep_origin      4206..4661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4293..4312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4503..4524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4678..4697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4726..4749)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5123..5142
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5212..5259
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        5251..5269
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5273..5282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5279..6082
                     /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(5342..5361)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5952..5971
                     /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 caaaatactg acgtagaaag taataatttc ttgggtagtt
      901 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      961 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgctg cagatacctg
     1021 cagagttgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     1081 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctcgagtact aggatccatt
     1141 aggctttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     1201 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     1261 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     1321 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     1381 caccgcttaa tcatgtctgt ttaccgttta agagctatgc tggaaacagc atagcaagtt
     1441 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct
     1501 cgagtactag gatccattag gctttcccat gattccttca tatttgcata tacgatacaa
     1561 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1621 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1681 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1741 cttgtggaaa ggacgaaaca ccgtaaaata acagactact ccggtttaag agctatgctg
     1801 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     1861 agtcggtgct ttttttctcg agtactagga tccattaggc tttcccatga ttccttcata
     1921 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1981 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2041 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2101 tttcttggct ttatatatct tgtggaaagg acgaaacacc gttttttttc tagaggtacc
     2161 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     2221 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2281 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2341 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
     2401 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     2461 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     2521 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggg ggggcgcgsg
     2581 ssaggsgggg gggggsgggg sgaggggsgg ggsggggsga ggsggagagg tgcggcggca
     2641 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
     2701 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
     2761 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
     2821 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
     2881 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
     2941 gaaatcactt tttttcaggt tggaccggtg ccaccatgag cgagctgatt aaggagaaca
     3001 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     3061 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     3121 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     3181 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     3241 catgggagag agtcaccaca tacgatgacg ggggcgtgct gaccgctacc caggacacca
     3301 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     3361 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     3421 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     3481 atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga
     3541 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaaa
     3601 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     3661 ggcacaagct taattaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag
     3721 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3781 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3841 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     3901 caggcatgct ggggagctct cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc
     3961 cccgggcgcg tactccacct cacccatcgc aggaacccct agtgatggag ttggccactc
     4021 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4081 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4141 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4201 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4261 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4501 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     4561 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     4621 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4681 aatctgctct gatgccgcat agtgcttttc ccaaggcagt ctggagcatg cgctttagca
     4741 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     4801 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     4861 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     4921 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     4981 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     5041 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     5101 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     5161 ttctcctctt cctcatctcc gggcctttcg accgatcatc aagctgcacg tgttgacaat
     5221 taatcatcgg catagtatat cggcatagta taatacgact cactatagga gggccaccat
     5281 gggatcggcc attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     5341 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     5401 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     5461 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     5521 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     5581 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     5641 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     5701 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5761 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     5821 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5881 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5941 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     6001 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     6061 ccttcttgac gagttcttct gatcgagaaa ttgatgatct attaaacaat aaagacgtcc
     6121 acatggaagt gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6181 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6241 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     6301 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6361 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6421 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6481 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6541 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
