LOCUS       Exported                7240 bp ds-DNA     circular SYN 28-FEB-2025
DEFINITION  PiggyBac plasmid for mU6-driven expression of a gRNA with enhanced 
            backbone. tagBFP and Puro resistance is driven driven by Ef1a. Clone
            new gRNA into BstXI and BlpI sites, replacing the stock GFP gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7240)
  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;56:1168-1180
  PUBMED    38724747
REFERENCE   2  (bases 1 to 7240)
  AUTHORS   Hackett Lab
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Feb 28, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7240
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    656..965
                     /label='Piggy 5'
     primer_bind     953..992
                     /label=p133_Cloning_primer_fw
     promoter        1014..1327
                     /label=mU6_promoter
     protein_bind    1208..1241
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    1328..1347
                     /label=gRNA protospacer (GFP)
     misc_feature    1348..1359
                     /label=modified stem loop
     misc_feature    1361..1372
                     /label=modified stem loop (1)
     misc_feature    1374..1440
                     /label=modified stem loop (2)
     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"
     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"
     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    4071..4104
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4124..4163)
                     /label=p133_cloning primer_rv
     polyA_signal    4211..4266
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4625..4641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4649..4665
                     /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(4673..4703)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4718..4739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    4798..5032
                     /label=Piggy 3'
     promoter        complement(5052..5070)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5091..5107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5115..5131
                     /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(5139..5169)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5184..5205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5493..6081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6252..7112)
                     /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"
     promoter        complement(7113..7217)
                     /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 gcggtaagtg 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 tgccatatgc tggctgccat gaacaaaggt ggctataaag
     4381 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     4441 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4501 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4561 catagctgtc cctcttctct tatgaagatc cctcgacctg cagcccaagc ttggcgtaat
     4621 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4681 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4741 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccttt
     4801 gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa taaacataaa
     4861 taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa acttaatatc
     4921 tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc gtcaatttta
     4981 cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt aaagagctcc
     5041 agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg
     5101 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5161 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5221 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5281 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5341 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5401 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5461 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5521 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5581 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5641 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5701 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5761 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5821 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5881 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5941 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6001 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6061 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6121 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6181 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6241 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6301 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6361 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6421 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6481 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6541 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6601 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6661 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6721 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6781 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6841 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6901 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6961 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7021 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7081 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7141 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7201 caaatagggg ttccgcgcac atttccccga aaagtgccac
//
