LOCUS       sequence-452250-        7731 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 7731)
  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 7731)
  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-14kb-USF-7-12
FEATURES             Location/Qualifiers
     source          1..7731
                     /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          3866..4093
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4111..4812
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4837..4854)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4843..5050
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5054..5123
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   5126..5266
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5126..5255
                     /label=AAV2 ITR
     rep_origin      5341..5796
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5428..5447)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5638..5659
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5813..5832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5861..5884)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6258..6277
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        6347..6394
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6386..6404
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6408..6417
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6414..7217
                     /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(6477..6496)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7087..7106
                     /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 aaacaccgag gtttaaaaga
     1021 agagtatagt 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 acaccggcac atggtaaagc gctagggttt 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 accgcagaaa ttagcccatg ttccgtttaa 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 cgatggtacc aagggcgagt
     2161 cagtttaaga 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 atgaagtcgt acctctaact 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 aaacaccgaa tcttagcatg aactgggggt 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 tccccaccct ccccaccccc aattttgtat ttatttattt
     3661 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggggggsc ssgsgsgggg
     3721 gggggggggg gggggggggg gggggggsgg ggsgrggcgg rgaggtgcgg cggcagccaa
     3781 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
     3841 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
     3901 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     3961 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
     4021 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
     4081 cacttttttt caggttggac cggtgccacc atgagcgagc tgattaagga gaacatgcac
     4141 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     4201 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     4261 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     4321 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     4381 gagagagtca ccacatacga tgacgggggc gtgctgaccg ctacccagga caccagcctc
     4441 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     4501 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     4561 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     4621 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     4681 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgaaacctac
     4741 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     4801 aagcttaatt aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     4861 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4921 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4981 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     5041 atgctgggga gctctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg
     5101 gcgcgtactc cacctcaccc atcgcaggaa cccctagtga tggagttggc cactccctct
     5161 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     5221 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     5281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     5341 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5401 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5461 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5521 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     5581 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5641 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     5701 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     5761 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     5821 gctctgatgc cgcatagtgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     5881 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     5941 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     6001 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     6061 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     6121 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     6181 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     6241 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     6301 ctcttcctca tctccgggcc tttcgaccga tcatcaagct gcacgtgttg acaattaatc
     6361 atcggcatag tatatcggca tagtataata cgactcacta taggagggcc accatgggat
     6421 cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     6481 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     6541 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     6601 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     6661 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     6721 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     6781 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     6841 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     6901 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     6961 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     7021 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     7081 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     7141 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     7201 ttgacgagtt cttctgatcg agaaattgat gatctattaa acaataaaga cgtccacatg
     7261 gaagtgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     7321 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     7381 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     7441 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     7501 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     7561 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     7621 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7681 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
