LOCUS       sequence-452315-        5557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  non-targeting control guide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5557)
  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 5557)
  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-NT-1mer-gRNA
FEATURES             Location/Qualifiers
     source          1..5557
                     /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"
     promoter        667..915
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     667..687
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     838..857
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        937..1012
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1125..1410
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1412..1691
                     /label=chicken beta-actin promoter
     intron          1692..1919
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1937..2638
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(2663..2680)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2669..2876
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2880..2949
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   2952..3092
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2952..3081
                     /label=AAV2 ITR
     rep_origin      3167..3622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3254..3273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3464..3485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3639..3658)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3687..3710)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4084..4103
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        4173..4220
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        4212..4230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4234..4243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4240..5043
                     /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(4303..4322)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4913..4932
                     /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 ccctggagac
      661 ccatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      721 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      781 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      841 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      901 aaaggacgaa acaccgccat tggcaggctc ctaccagttt tagagctaga aatagcaagt
      961 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
     1021 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
     1081 tctgcagaca aatggctcta gaggtctccc tatctagagg tacccgttac ataacttacg
     1141 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg
     1201 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     1261 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1321 tggcccgcct ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac
     1381 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
     1441 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
     1501 tgtgcagcga tgggggcggg gggggggggg gggggcgcgc gcsagggggg gggggggggg
     1561 gsgaggggsg gggsggggsg aggsggagag gtgcggcggc agccaatcag agcggcgcgc
     1621 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
     1681 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
     1741 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
     1801 gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg
     1861 ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg
     1921 ttggaccggt gccaccatga gcgagctgat taaggagaac atgcacatga agctgtacat
     1981 ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta
     2041 cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     2101 cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg
     2161 catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac
     2221 atacgatgac gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct
     2281 catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa
     2341 gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg gcggcctgga
     2401 aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa
     2461 gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt
     2521 ggactacaga ctggaaagaa tcaaggaggc caacaacgaa acctacgtcg agcagcacga
     2581 ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc ttaattaaga
     2641 attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2701 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2761 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2821 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagctc
     2881 tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc
     2941 tcacccatcg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3001 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3061 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     3121 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3181 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3241 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3301 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3361 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     3421 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3481 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     3541 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3601 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     3661 tagtgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     3721 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     3781 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3841 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     3901 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     3961 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     4021 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     4081 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     4141 cgggcctttc gaccgatcat caagctgcac gtgttgacaa ttaatcatcg gcatagtata
     4201 tcggcatagt ataatacgac tcactatagg agggccacca tgggatcggc cattgaacaa
     4261 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4321 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4381 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4441 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4501 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4561 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4621 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4681 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4741 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4801 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4861 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4921 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4981 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5041 tgatcgagaa attgatgatc tattaaacaa taaagacgtc cacatggaag tgcaaactat
     5101 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5161 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5221 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     5281 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5341 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5401 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5461 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5521 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
