LOCUS       sequence-470313-        7242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enhanced guide RNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  AUTHORS   Policarpi C, Munafo M, Tsagkris S, Carlini V, Hackett JA
  TITLE     Systematic epigenome editing captures the context-dependent 
            instructive function of chromatin modifications.
  JOURNAL   Nat Genet. 2024 Jun;56(6):1168-1180. doi: 
            10.1038/s41588-024-01706-w. Epub 2024 May 9.
  PUBMED    38724747
REFERENCE   2  (bases 1 to 7242)
  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: 
            "10.1038/s41588-024-01706-w"; journalName: "Nat Genet"; date: 
            "2024-06"; volume: "56"; issue: "6"; pages: "1168-1180"
COMMENT     Sequence Label: pPB_mU6_enh-gRNA_Puro-T2A-BFP
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    complement(1208..1241)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1245..1265
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1510..1721
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1722..2660
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2617..2637
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2681..3277
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2681..2700)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3177..3197
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3287..3340
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3353..4051
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(4071..4104)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4146..4165)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4211..4266
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4265..4284)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4627..4643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4651..4667
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4675..4681,4682..4699,4700..4705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4720..4741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4968..5030
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5054..5072)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5093..5109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5117..5133
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5141..5147,5148..5165,5166..5171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5186..5207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5324..5341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5495..6083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5575..5594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6254..7045,7046..7114))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6877..6896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7115..7219)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatatcgt cgaagcagag atccagtttg gttagtaccg ggcccgctct agagatccga
     1021 cgcgccatct ctaggcccgc gccggccccc tcgcacggac ttgtgggaga agctcggcta
     1081 ctcccctgcc ccggttaatt tgcatataat atttcctagt aactatagag gcttaatgtg
     1141 cgataaaaga cagataatct gttcttttta atactagcta cattttacat gataggcttg
     1201 gatttctata acttcgtata gcatacatta tacgaagtta taaacagcac aaaaggaaac
     1261 tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata agtatccctt ggagaaccac
     1321 cttgttggac caggatgggc accacccgtt taagagctaa gctggaaaca gcatagcaag
     1381 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     1441 ctcgagtact aggatccatt aggcggccgc gtggataacc gtattaccgc catgcatgtg
     1501 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     1561 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     1621 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     1681 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
     1741 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
     1801 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     1861 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
     1921 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     1981 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     2041 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     2101 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     2161 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     2221 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     2281 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     2341 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     2401 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcgc
     2461 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     2521 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     2581 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     2641 gtttttttct tccatttcag gtgtcgtgag ctagcccacc atgaccgagt acaagcccac
     2701 ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt
     2761 cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac
     2821 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     2881 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     2941 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3001 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3061 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3121 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3181 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3241 accgcgcacc tggtgcatga cccgcaagcc cggtgccgga tcgggagagg gcagaggaag
     3301 tctgctaaca tgcggtgacg tcgaggagaa tcctggccca ccggtcgcca ccagcgagct
     3361 gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt
     3421 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa
     3481 ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta
     3541 cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt
     3601 ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc
     3661 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt
     3721 gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac
     3781 cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct
     3841 cgtgggcggg agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc
     3901 taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga
     3961 ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct
     4021 ccctagcaaa ctggggcaca agcttaatta agaattcgtc gagggaccta ataacttcgt
     4081 atagcataca ttatacgaag ttatacatgt ttaagggttc cggttccact aggtacaatt
     4141 cgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     4201 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     4261 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     4321 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     4381 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     4441 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     4501 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     4561 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     4621 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4681 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4741 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcct
     4801 ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata
     4861 aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata
     4921 tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt
     4981 tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg ttaaagagct
     5041 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     5101 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5161 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5221 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5281 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5341 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6241 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6301 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6361 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6421 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6481 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6541 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6601 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6661 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6721 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6781 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6841 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6901 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6961 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7021 ttactttcac cagcgtttct gggtgagcaa aaacgggaag gcaaaatgcc gcaaaaaagg
     7081 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7141 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7201 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
