LOCUS       sequence-452294-        7349 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 7349)
  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 7349)
  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-35kb-DSF
FEATURES             Location/Qualifiers
     source          1..7349
                     /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..3483
                     /label=chicken beta-actin promoter
     intron          3484..3711
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3729..4430
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4455..4472)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4461..4668
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4672..4741
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   4744..4884
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4744..4873
                     /label=AAV2 ITR
     rep_origin      4959..5414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5046..5065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5256..5277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5431..5450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5479..5502)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5876..5895
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5965..6012
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        6004..6022
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6026..6035
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6032..6835
                     /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(6095..6114)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6705..6724
                     /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 aaacaccgct gcagatacct
     1021 gcagagttgt 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 acaccgctta atcatgtctg tttaccgttt 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 accgtaaaat aacagactac tccggtttaa 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 cgtttatatt cattcaatta
     2161 gcgtttaaga 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 tatttcctcg tcccctctgc 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 gggggggcgc gcgcsaggsg ggggggggsg gggsgagggg
     3361 sggggsgggg sgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     3421 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     3481 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     3541 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     3601 cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg
     3661 gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg
     3721 gtgccaccat gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
     3781 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     3841 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     3901 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
     3961 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgatg
     4021 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     4081 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
     4141 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
     4201 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat
     4261 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     4321 gactggaaag aatcaaggag gccaacaacg aaacctacgt cgagcagcac gaggtggcag
     4381 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattaa gaattcctag
     4441 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4501 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4561 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4621 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc tctcgaagcc
     4681 gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca cctcacccat
     4741 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4801 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4861 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4921 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4981 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     5041 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5101 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5161 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5221 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5281 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     5341 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5401 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagtgctt
     5461 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca
     5521 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc
     5581 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc
     5641 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg
     5701 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag
     5761 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg
     5821 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     5881 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     5941 tcgaccgatc atcaagctgc acgtgttgac aattaatcat cggcatagta tatcggcata
     6001 gtataatacg actcactata ggagggccac catgggatcg gccattgaac aagatggatt
     6061 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     6121 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     6181 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     6241 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     6301 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     6361 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     6421 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     6481 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     6541 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     6601 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     6661 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     6721 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     6781 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgatcgag
     6841 aaattgatga tctattaaac aataaagacg tccacatgga agtgcaaact attaactggc
     6901 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6961 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7021 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7081 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7141 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7201 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7321 gaccccgtag aaaagatcaa aggatcttc
//
