LOCUS       sequence-452286-        7727 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 7727)
  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 7727)
  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-42kb-USF
FEATURES             Location/Qualifiers
     source          1..7727
                     /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"
     promoter        3583..3861
                     /label=chicken beta-actin promoter
     intron          3862..4089
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4107..4808
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4833..4850)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4839..5046
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5050..5119
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   5122..5262
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5122..5251
                     /label=AAV2 ITR
     rep_origin      5337..5792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5424..5443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5634..5655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5809..5828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5857..5880)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6254..6273
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        6343..6390
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6382..6400
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6404..6413
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6410..7213
                     /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(6473..6492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7083..7102
                     /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 aaacaccgtt tcaacgtgta
     1021 ttattaaagt 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 acaccgcaag ggccataaat cagtatgttt 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 accgatggac cagcatacac tgtcgtttaa 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 cgctggattt gccacccaca
     2161 tagtttaaga 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 aggcccagga gtatatgaac 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 aaacaccgta gcacaaagca ccatcggtgt 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 gggggcgcgc gcsagggggg
     3721 gsgggggggg gsgaggggsg gggsggggsg aggcggagag gtgcggcggc agccaatcag
     3781 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     3841 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     3901 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     3961 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
     4021 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
     4081 ttttttcagg ttggaccggt gccaccatga gcgagctgat taaggagaac atgcacatga
     4141 agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag
     4201 gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc
     4261 ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc
     4321 acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga
     4381 gagtcaccac atacgatgac gggggcgtgc tgaccgctac ccaggacacc agcctccagg
     4441 acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg
     4501 tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg
     4561 gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg
     4621 caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg
     4681 tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgaa acctacgtcg
     4741 agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc
     4801 ttaattaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     4861 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4921 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4981 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     5041 tggggagctc tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc
     5101 gtactccacc tcacccatcg caggaacccc tagtgatgga gttggccact ccctctctgc
     5161 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     5221 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     5281 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     5341 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5401 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5461 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5521 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     5581 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5641 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     5701 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5761 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5821 tgatgccgca tagtgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
     5881 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
     5941 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     6001 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     6061 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     6121 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     6181 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     6241 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     6301 tcctcatctc cgggcctttc gaccgatcat caagctgcac gtgttgacaa ttaatcatcg
     6361 gcatagtata tcggcatagt ataatacgac tcactatagg agggccacca tgggatcggc
     6421 cattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     6481 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     6541 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     6601 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     6661 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     6721 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     6781 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     6841 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     6901 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     6961 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     7021 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     7081 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     7141 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     7201 cgagttcttc tgatcgagaa attgatgatc tattaaacaa taaagacgtc cacatggaag
     7261 tgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     7321 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     7381 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     7441 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7501 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7561 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7621 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
