LOCUS       sequence-452292-        7350 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 7350)
  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 7350)
  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-Fgf5Pro
FEATURES             Location/Qualifiers
     source          1..7350
                     /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"
     promoter        3205..3484
                     /label=chicken beta-actin promoter
     intron          3485..3712
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3730..4431
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4456..4473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4462..4669
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4673..4742
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   4745..4885
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4745..4874
                     /label=AAV2 ITR
     rep_origin      4960..5415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5047..5066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5257..5278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5432..5451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5480..5503)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5877..5896
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5966..6013
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6005..6023
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6027..6036
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6033..6836
                     /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(6096..6115)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6706..6725
                     /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 aaacaccggg gtggctcttg
     1021 acaaccgcgt 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 acaccgggtc gcagccagcg tcccgggttt 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 accggatgcg atcccgggag tggggtttaa 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 cgcacccccg cccccgggac
     2161 cggtttaaga 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 ccgctttgtg tactcacggc 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 ggggggggcg cgcgcsaggg gggggggggg ggggsgaggg
     3361 gsggggsggg gsgaggsgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
     3421 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     3481 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     3541 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     3601 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
     3661 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
     3721 ggtgccacca tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     3781 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     3841 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     3901 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     3961 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgat
     4021 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     4081 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     4141 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     4201 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
     4261 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     4321 agactggaaa gaatcaagga ggccaacaac gaaacctacg tcgagcagca cgaggtggca
     4381 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta agaattccta
     4441 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4501 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4561 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4621 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag ctctcgaagc
     4681 cgcggtgcgg gtgccagggc gtgcccttgg gctccccggg cgcgtactcc acctcaccca
     4741 tcgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4801 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4861 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4921 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4981 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     5041 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     5101 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     5161 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5221 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5281 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5341 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5401 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagtgct
     5461 tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac
     5521 aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt
     5581 ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc
     5641 cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt
     5701 gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata
     5761 gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg
     5821 gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     5881 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     5941 ttcgaccgat catcaagctg cacgtgttga caattaatca tcggcatagt atatcggcat
     6001 agtataatac gactcactat aggagggcca ccatgggatc ggccattgaa caagatggat
     6061 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     6121 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     6181 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     6241 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     6301 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     6361 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     6421 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     6481 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     6541 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     6601 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     6661 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     6721 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     6781 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgatcga
     6841 gaaattgatg atctattaaa caataaagac gtccacatgg aagtgcaaac tattaactgg
     6901 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6961 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7021 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7081 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7141 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7201 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7261 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7321 agaccccgta gaaaagatca aaggatcttc
//
