LOCUS       sequence-452244-        7729 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 7729)
  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 7729)
  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-25kb-USF
FEATURES             Location/Qualifiers
     source          1..7729
                     /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          3864..4091
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4109..4810
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4835..4852)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4841..5048
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5052..5121
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   5124..5264
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5124..5253
                     /label=AAV2 ITR
     rep_origin      5339..5794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5426..5445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5636..5657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5811..5830)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5859..5882)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6256..6275
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        6345..6392
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6384..6402
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6406..6415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6412..7215
                     /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(6475..6494)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7085..7104
                     /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 aaacaccgga cgttagatgc
     1021 ctgaactagt 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 acaccgtcag cagttatggg tacctagttt 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 accgaaatac tgagtgttac acacgtttaa 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 cgctgcaact taattctccc
     2161 ttgtttaaga 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 caaattttct catgcaacga 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 aaacaccgtg aattagaatg tcttggcagt 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 gggggggcgc gcgssagggg
     3721 gggggggggg gggsgagggg sggggsgggg sgaggsggag aggtgcggcg gcagccaatc
     3781 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     3841 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     3901 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     3961 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     4021 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     4081 ctttttttca ggttggaccg gtgccaccat gagcgagctg attaaggaga acatgcacat
     4141 gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga
     4201 aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct
     4261 ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa
     4321 ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga
     4381 gagagtcacc acatacgatg acgggggcgt gctgaccgct acccaggaca ccagcctcca
     4441 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc
     4501 tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga
     4561 cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat
     4621 cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg
     4681 cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg aaacctacgt
     4741 cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa
     4801 gcttaattaa gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     4861 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4921 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4981 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     5041 gctggggagc tctcgaagcc gcggtgcggg tgccagggcg tgcccttggg ctccccgggc
     5101 gcgtactcca cctcacccat cgcaggaacc cctagtgatg gagttggcca ctccctctct
     5161 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     5221 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     5281 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     5341 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5401 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5461 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5521 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     5581 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5641 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     5701 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5761 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     5821 tctgatgccg catagtgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     5881 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta
     5941 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag
     6001 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag
     6061 cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc
     6121 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa
     6181 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt
     6241 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     6301 cttcctcatc tccgggcctt tcgaccgatc atcaagctgc acgtgttgac aattaatcat
     6361 cggcatagta tatcggcata gtataatacg actcactata ggagggccac catgggatcg
     6421 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     6481 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     6541 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     6601 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     6661 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     6721 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     6781 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     6841 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     6901 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     6961 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     7021 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     7081 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     7141 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     7201 gacgagttct tctgatcgag aaattgatga tctattaaac aataaagacg tccacatgga
     7261 agtgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7321 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7381 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7441 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7501 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7561 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7621 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7681 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
