LOCUS       sequence-452326-        5552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for CARGO cloning, with U6 promoter and gRNA scaffold, 
            TagBFP, and Kanamycin resistence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5552)
  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 5552)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..5552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        456..741
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        743..1020
                     /label=chicken beta-actin promoter
     intron          1021..1248
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1266..1967
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYDDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1992..2009)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1998..2205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2209..2278
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   2281..2421
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2281..2410
                     /label=AAV2 ITR
     rep_origin      2496..2951
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2583..2602)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2793..2814
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2968..2987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3016..3039)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3413..3432
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3502..3549
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        3541..3559
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3563..3572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3569..4372
                     /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(3632..3651)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4242..4261
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      4887..5475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5376..5395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtctcc ctatctagag gtacccgtta cataacttac ggtaaatggc
      481 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg
      541 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      601 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      661 tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      721 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
      781 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      841 atgggggcgg gggggggggg ggggcgcgcg ccaggsgggg sggggggggg sgaggggsgg
      901 ggsggggsga ggsggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      961 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1021 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1081 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1141 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     1201 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     1261 ccaccatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     1321 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     1381 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     1441 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
     1501 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgatgacg
     1561 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     1621 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     1681 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     1741 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
     1801 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     1861 tggaaagaat caaggaggcc aacaacgaaa cctacgtcga gcagcacgag gtggcagtgg
     1921 ccagatactg cgacctccct agcaaactgg ggcacaagct taattaagaa ttcctagagc
     1981 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2041 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2101 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2161 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagctct cgaagccgcg
     2221 gtgcgggtgc cagggcgtgc ccttgggctc cccgggcgcg tactccacct cacccatcgc
     2281 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2341 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2401 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     2461 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     2521 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     2581 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2641 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2701 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2761 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2821 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     2881 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2941 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agtgcttttc
     3001 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     3061 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     3121 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     3181 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     3241 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     3301 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     3361 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     3421 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     3481 accgatcatc aagctgcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     3541 taatacgact cactatagga gggccaccat gggatcggcc attgaacaag atggattgca
     3601 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3661 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3721 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3781 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3841 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3901 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3961 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4021 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4081 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4141 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4201 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4261 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4321 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gatcgagaaa
     4381 ttgatgatct attaaacaat aaagacgtcc acatggaagt gcaaactatt aactggcgaa
     4441 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4501 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4561 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4621 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4681 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4741 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4801 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4861 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4921 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4981 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5041 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5101 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5161 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5221 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5281 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5341 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5401 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5461 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5521 ccttttgctc acatgtccct ggagacccat gt
//
