LOCUS       Exported                7493 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac transposon system construct with 5 concatenated U6 promoter
            driven transcriptional cassettes for the activation of OCT4. 
            Contains PGK-puro selection cassette..
ACCESSION   .
VERSION     .
KEYWORDS    PB-GG-OCT4-1-5-PGK-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7493
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    complement(118..151)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        212..711
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(247..270)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     644..663
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             751..1350
                     /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(751..770)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(1431..1448)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1437..1661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1708..1724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1732..1748
                     /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(1756..1786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1801..1822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1881..2005)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   2295..2329
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2358..2377)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2359..2377)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2387..2403)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2396..2418)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2545..3000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2632..2651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2842..2863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3026..3130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3131..3991
                     /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(3349..3368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4162..4750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4651..4670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4904..4921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5038..5059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5074..5104
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5112..5128
                     /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     5136..5152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5171..5191
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5173..5191
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   5242..5304
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     5517..5533
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5534..5774
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        5803..5878
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5910..5926
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5927..6167
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        6197..6272
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6284..6300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6304..6320
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6321..6561
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6697..6713
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6714..6954
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        6984..7059
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     7091..7107
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7108..7348
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        7378..7453
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
       61 ccgggcgacc aaaggtcgcc cgacgcccaa ttccgatcat attcaataac ccttaatata
      121 acttcgtata atgtatgcta tacgaagtta ttaggtctga agaggagttt acgtccagcc
      181 aagcttagga tctcgacctc gaaattctac cgggtagggg aggcgctttt cccaaggcag
      241 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
      301 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
      361 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
      421 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
      481 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
      541 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
      601 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
      661 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcatc
      721 catctagatc tcgagcagct gaagcttacc atgaccgagt acaagcccac ggtgcgcctc
      781 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
      841 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
      901 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
      961 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     1021 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     1081 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     1141 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     1201 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccacaa cctccccttc
     1261 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     1321 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     1381 gaaaggagcg cacgacccca tgcatcgatg atctagagct cgctgatcag cctcgactgt
     1441 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1501 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1561 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1621 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1681 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1741 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     1801 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1861 cctgtcgtgc cagcggatcc catagtgact ggatatgttg tgttttacag tattatgtag
     1921 tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc
     1981 gttcagcttt tttgtacaaa cttgtgctag cattctagtt gatatctata acaagaaaat
     2041 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga
     2101 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt
     2161 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc
     2221 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca
     2281 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taatctagaa
     2341 tggcgcgccc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     2401 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2461 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2521 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2581 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2641 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2701 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2761 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2821 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2881 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2941 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3001 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3061 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3361 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3661 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4921 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4981 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5041 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5101 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5161 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggattaat taagcggccg
     5221 catacgcgta tactagatta accctagaaa gataatcata ttgtgacgta cgttaaagat
     5281 aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta
     5341 atttgaatag atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca
     5401 aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta
     5461 taaagtaaca aaacttttaa ctagaggatc tcgagctcaa gcttcgaatt ccccctgtaa
     5521 aacgacggcc agtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     5581 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     5641 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     5701 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     5761 cttgtggaaa ggacgaaaca ccggggggag aaactgaggc gagttttaga gctagaaata
     5821 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     5881 tttttcatgg tcatagctgt ttcctcatgg taaaacgacg gccagtgagg gcctatttcc
     5941 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     6001 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     6061 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     6121 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtg
     6181 gtggcaatgg tgtctggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     6241 atcaacttga aaaagtggca ccgagtcggt gctttttttc atggtcatag ctgtttcctg
     6301 gacgtaaaac gacggccagt gagggcctat ttcccatgat tccttcatat ttgcatatac
     6361 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     6421 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     6481 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     6541 tatatatctt gtggaaagga cgaaacaccg gacacaactg gcgcccctcc gttttagagc
     6601 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     6661 cgtgcttttt ttcatggtca tagctgtttc ctccaggtaa aacgacggcc agtgagggcc
     6721 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     6781 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     6841 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     6901 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     6961 ccgggcacag tgccagaggt ctggttttag agctagaaat agcaagttaa aataaggcta
     7021 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttcatg gtcatagctg
     7081 tttccttgtt gtaaaacgac ggccagtgag ggcctatttc ccatgattcc ttcatatttg
     7141 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     7201 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     7261 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     7321 ttggctttat atatcttgtg gaaaggacga aacaccgtct gtgggggacc tgcactggtt
     7381 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     7441 accgagtcgg tgcttttttt catggtcata gctgtttcct aacgagcggc cgc
//
