LOCUS       sequence-194747-        5943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon system construct for U6 promoter-driven 
            expression of a gRNA targeting EEA-motif. Includes PGK-puro 
            selection cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5943)
  AUTHORS   Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti
            EM, Trokovic R, Kere J, Otonkoski T
  TITLE     Human pluripotent reprogramming with CRISPR activators.
  JOURNAL   Nat Commun. 2018 Jul 6;9(1):2643. doi: 10.1038/s41467-018-05067-x.
  PUBMED    29980666
REFERENCE   2  (bases 1 to 5943)
  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"; journalName: "Nat 
            Commun."; date: "2018-07-6"; volume: "9(1):2643. doi"; pages: " 
            10.1038/s41467-018-05067-x"
COMMENT     Sequence Label: PB-EEA-g1-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..5943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..59
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        60..308
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     60..80
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     231..250
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        324..399
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    557..590
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        651..1148
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(686..709)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1081..1100
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1188..1787
                     /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(1188..1207)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1684..1704
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1868..1885)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1874..2098
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2145..2161)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2169..2185
                     /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(2193..2199,2200..2217,2218..2223))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2238..2259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(2318..2442)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   2732..2766
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2795..2814)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2796..2814)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2824..2840)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2833..2855)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2982..3437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3069..3088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3279..3300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3463..3567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3568..3636,3637..4428)
                     /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     complement(3786..3805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4599..5187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5088..5107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5475..5496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5511..5516,5517..5534,5535..5541)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5549..5565
                     /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)."
     primer_bind     5573..5589
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5608..5628
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5610..5628
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   5679..5741
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 tcgactctag aggatctcga gctcaagctt cgaattcccc ctgtaaaacg acggccagtg
       61 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      121 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      181 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      241 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      301 gaaacaccgc ccagcacttt ggggttttag agctagaaat agcaagttaa aataaggcta
      361 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttcatg gtcatagctg
      421 tttcctcatg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
      481 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccaat tccgatcata
      541 ttcaataacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctgaa
      601 gaggagttta cgtccagcca agcttaggat ctcgacctcg aaattctacc gggtagggga
      661 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg cacttggcgc
      721 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
      781 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
      841 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
      901 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
      961 caatagcagc tttgctcctt cgctttctgg gctcagcagc tgggaagggt gggtccgggg
     1021 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg
     1081 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg
     1141 gcctttcgac ctgcatccat ctagatctcg agcagctgaa gcttaccatg accgagtaca
     1201 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     1261 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     1321 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     1381 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     1441 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     1501 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     1561 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     1621 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     1681 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     1741 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     1801 acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcgatgatc tagagctcgc
     1861 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1921 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1981 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2041 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2101 tctgaggcgg aaagaacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
     2161 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2221 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2281 gctttccagt cgggaaacct gtcgtgccag cggatcccat agtgactgga tatgttgtgt
     2341 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     2401 atcattttac gtttctcgtt cagctttttt gtacaaactt gtgctagcat tctagttgat
     2461 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
     2521 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
     2581 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
     2641 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
     2701 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
     2761 ctagggttaa tctagaatgg cgcgccccaa ttcgccctat agtgagtcgt attacgcgcg
     2821 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     2881 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2941 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     3001 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3061 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3121 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3181 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3241 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3301 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3361 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3421 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3481 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3541 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3601 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3661 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3721 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3781 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3841 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3901 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3961 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4021 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4081 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4141 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4201 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4261 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4321 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4381 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4441 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4501 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4561 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4621 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4681 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4741 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4801 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4861 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4921 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4981 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5041 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5101 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5161 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5221 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5281 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5341 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     5401 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5461 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     5521 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     5581 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     5641 gattaattaa gcggccgcat acgcgtatac tagattaacc ctagaaagat aatcatattg
     5701 tgacgtacgt taaagataat catgcgtaaa attgacgcat gtgttttatc ggtctgtata
     5761 tcgaggttta tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac
     5821 taataataaa ttcaacaaac aatttattta tgtttattta tttattaaaa aaaaacaaaa
     5881 actcaaaatt tcttctataa agtaacaaaa cttttaacta gaggatctcg agctcaagct
     5941 tcg
//
