LOCUS       sequence-452234-        7730 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 7730)
  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 7730)
  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-1.9kb-USF
FEATURES             Location/Qualifiers
     source          1..7730
                     /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          3865..4092
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4110..4811
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4836..4853)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4842..5049
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    5053..5122
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   5125..5265
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5125..5254
                     /label=AAV2 ITR
     rep_origin      5340..5795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5427..5446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5637..5658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5812..5831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5860..5883)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6257..6276
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        6346..6393
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6385..6403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6407..6416
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6413..7216
                     /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(6476..6495)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7086..7105
                     /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 aaacaccgtg tgcccatagc
     1021 ttgcaccagt 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 acaccgagtg gctcataaat agcaacgttt 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 accgattcac cctaagtccc ctgcgtttaa 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 cggtatagag tcacactcat
     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 cagaggaaac tctctgttgg 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 aaacaccgct ctctcacaca tgactagcgt 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 ggggggggcg cgsgcsaggs
     3721 gggggggggg ggggsgaggg gsggggsggg gsgaggsgga gaggtgcggc ggcagccaat
     3781 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     3841 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     3901 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     3961 agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt
     4021 aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc
     4081 actttttttc aggttggacc ggtgccacca tgagcgagct gattaaggag aacatgcaca
     4141 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     4201 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     4261 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     4321 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     4381 agagagtcac cacatacgat gacgggggcg tgctgaccgc tacccaggac accagcctcc
     4441 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     4501 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     4561 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     4621 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     4681 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gaaacctacg
     4741 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     4801 agcttaatta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     4861 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4921 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4981 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     5041 tgctggggag ctctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg gctccccggg
     5101 cgcgtactcc acctcaccca tcgcaggaac ccctagtgat ggagttggcc actccctctc
     5161 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     5221 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     5281 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     5341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5401 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5521 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5581 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5641 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5701 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5761 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5821 ctctgatgcc gcatagtgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     5881 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     5941 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     6001 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     6061 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     6121 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     6181 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     6241 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     6301 tcttcctcat ctccgggcct ttcgaccgat catcaagctg cacgtgttga caattaatca
     6361 tcggcatagt atatcggcat agtataatac gactcactat aggagggcca ccatgggatc
     6421 ggccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     6481 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     6541 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     6601 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     6661 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     6721 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     6781 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     6841 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     6901 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     6961 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     7021 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     7081 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     7141 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     7201 tgacgagttc ttctgatcga gaaattgatg atctattaaa caataaagac gtccacatgg
     7261 aagtgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7321 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7381 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     7441 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7501 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7561 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7621 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7681 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
