LOCUS       sequence-198395-        5948 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 5948)
  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 5948)
  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..5948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   205..267
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     497..513
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        514..762
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     514..534
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     685..704
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        778..853
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1029..1062
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1123..1622
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1158..1181)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1555..1574
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1662..2261
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1662..1681)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2342..2359)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2348..2572
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2619..2635)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2643..2659
                     /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(2667..2673,2674..2691,2692..2697))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2712..2733
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(2792..2916)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   3206..3240
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3269..3288)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3270..3288)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3298..3314)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3307..3329)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3456..3911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3543..3562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3753..3774
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3937..4041
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4042..4110,4111..4902)
                     /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(4260..4279)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5073..5661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5562..5581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggatt aattaagcgg
      181 ccgcatacgc gtatactaga ttaaccctag aaagataatc atattgtgac gtacgttaaa
      241 gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta
      301 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
      361 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
      421 ctataaagta acaaaacttt taactagagg atctcgagct caagcttcga attcggatcc
      481 tcgagcgtct caccctgtaa aacgacggcc agtgagggcc tatttcccat gattccttca
      541 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      601 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      661 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      721 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcccagca ctttggggtt
      781 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      841 accgagtcgg tgcttttttt catggtcata gctgtttcct catgtgagac gagatctgtc
      901 gacgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
      961 gctcactgag gccgggcgac caaaggtcgc ccgacgccca attccgatca tattcaataa
     1021 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtctg aagaggagtt
     1081 tacgtccagc caagcttagg atctcgacct cgaaattcta ccgggtaggg gaggcgcttt
     1141 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     1201 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
     1261 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     1321 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     1381 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     1441 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     1501 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     1561 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     1621 cgacctgcat ccatctagat ctcgagcagc tgaagcttac catgaccgag tacaagccca
     1681 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     1741 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     1801 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     1861 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     1921 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     1981 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     2041 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     2101 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccaca
     2161 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     2221 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     2281 agcgcccgac cgaaaggagc gcacgacccc atgcatcgat gatctagagc tcgctgatca
     2341 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2401 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2461 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2521 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2581 gcggaaagaa cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga
     2641 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2701 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2761 cagtcgggaa acctgtcgtg ccagcggatc ccatagtgac tggatatgtt gtgttttaca
     2821 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     2881 ttacgtttct cgttcagctt ttttgtacaa acttgtgcta gcattctagt tgatatctat
     2941 aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa
     3001 ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc
     3061 acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg
     3121 gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga
     3181 aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg
     3241 ttaatctaga atggcgcgcc ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     3301 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3361 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3421 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     3481 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3541 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3601 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3661 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3721 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3781 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3841 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3901 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3961 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4021 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4081 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4141 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4201 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4261 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4321 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4381 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4441 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4501 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4561 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4621 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4681 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4741 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     4801 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4861 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4921 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4981 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5041 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5101 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5161 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5221 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5281 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5341 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5401 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5461 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5521 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5581 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5641 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5701 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5761 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5821 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5881 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5941 aacgcaat
//
