LOCUS       sequence-489406-        8338 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  A piggybac-based vector containing mouse U6 promoter-driven NANOG 
            sgRNA and CAG promoter-driven nuclear-localized Clover-T2A-BSR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8338)
  AUTHORS   Okubo C, Nakamura M, Sato M, Shichino Y, Mito M, Takashima Y, 
            Iwasaki S, Takahashi K
  TITLE     EIF3D safeguards the homeostasis of key signaling pathways in human 
            primed pluripotency.
  JOURNAL   Sci Adv. 2025 Apr 11;11(15):eadq5484. doi: 10.1126/sciadv.adq5484. 
            Epub 2025 Apr 9.
  PUBMED    40203091
REFERENCE   2  (bases 1 to 8338)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2025-04-11"; volume: "11(15):eadq5484. doi"; pages: " 
            10.1126/sciadv.adq5484. Epub 2025 Apr 9"
COMMENT     Sequence Label: PB-U6-CNCB_sgNANOG
FEATURES             Location/Qualifiers
     source          1..8338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    complement(1579..1612)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1616..1636
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        1956..2259
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2261..2536
                     /label=chicken beta-actin promoter
     intron          2537..3553
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3477..3499
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3561..3580
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3618..3638
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3642..3662
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3666..3686
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3702..3704,3705..3707,3708..4418)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3747..3768)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4355..4376
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4419..4472
                     /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"
     polyA_signal    4908..5029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4945..4964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4999..5018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5792..5826
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6317..6333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6317..6333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6330..6352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6341..6357
                     /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(6365..6371,6372..6389,6390..6395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6410..6431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6548..6565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6719..7307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6799..6818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7478..8269,8270..8338))
                     /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     8101..8120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggcccgat gaggcccttt cgtcttcact
     1381 cgaggatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacgga cttgtgggag
     1441 aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag taactataga
     1501 ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct acattttaca
     1561 tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt ataaacagca
     1621 caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aagtatccct
     1681 tggagaacca ccttgttggc agcagaacgt taaaatccgt ttaagagcta tgctggaaac
     1741 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1801 gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg
     1861 ccatgcatcc cgggtgactg actgagcggc cgcactagtt attaatagta atcaattacg
     1921 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1981 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     2041 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     2101 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     2161 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     2221 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     2281 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     2341 taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg
     2401 gsggggsgag gggsggggsg gggsgaggsg gagaggtgsg gcggcagcca atcagagcgg
     2461 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     2521 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     2581 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     2641 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     2701 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
     2761 ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc
     2821 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga
     2881 ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg
     2941 ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct
     3001 gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg
     3061 gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggkggg
     3121 ggtgccgggc ggggcggggc cgcckcgggc cggggrgggc tcgggggrgg ggcgcggcgg
     3181 cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta
     3241 atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg
     3301 gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa
     3361 ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca
     3421 gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
     3481 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
     3541 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
     3601 agaattccgc caccatgcca aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg
     3661 tagatccaaa aaagaagaga aaggtacacg tgtcaacgcg tatggtgagc aagggcgagg
     3721 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3781 agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt
     3841 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct
     3901 acggcgtggc ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3961 ccgccatgcc cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggtacct
     4021 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4081 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca
     4141 acagccacaa cgtctatatc acggccgaca agcagaagaa cggcatcaag gctaacttca
     4201 agatccgcca caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4261 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtccg
     4321 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4381 ccgccgggat tacacatggc atggacgagc tgtacaagga gggcagagga agtcttctaa
     4441 catgcggtga cgtggaggag aatcccggcc ctatgaagac cttcaacatc tcccagcagg
     4501 atctagaatt agtagaagta gcgacagaga agattacaat gctttatgag gataataaac
     4561 atcatgtggg agcggcaatt cgtacgaaaa caggagaaat catttcggca gtacatattg
     4621 aagcgtatat aggacgagta actgtttgtg cagaagccat tgcgattggt agtgcagttt
     4681 cgaatggaca aaaggatttt gacacgattg tagctgttag acacccttat tctgacgaag
     4741 tagatagaag tattcgagtg gtaagtcctt gtggtatgtg tagggagttg atttcagact
     4801 atgcaccaga ttgttttgtg ttaatagaaa tgaatggcaa gttagtcaaa actacgattg
     4861 aagaactcat tccactcaaa tatacccgaa attaagttaa ctaactaaac ttgtttattg
     4921 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4981 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     5041 attgactcaa atgatgtcaa ttagtctatc agaagctcat ctggtctccc ttccggggga
     5101 caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc ttatatccct
     5161 tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt ccctaaagaa
     5221 acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg gcccattggt
     5281 atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt
     5341 cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg
     5401 ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc
     5461 cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccctgat
     5521 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
     5581 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
     5641 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
     5701 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
     5761 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
     5821 ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg gctcagtcat
     5881 cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt ttcccgcgag
     5941 gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga cgttgagcga
     6001 aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt taacggtgaa
     6061 ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac acagttccgg
     6121 atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg gaactgccgt
     6181 gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag cgaggacggg
     6241 tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc cggcgggcgc
     6301 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     6361 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     6421 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6481 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6541 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6601 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6661 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6721 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6781 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6841 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6901 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6961 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     7021 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     7081 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     7141 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     7201 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     7261 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     7321 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7381 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7441 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7501 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7561 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7621 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7681 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7741 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7801 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7861 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7921 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7981 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     8041 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     8101 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     8161 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     8221 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     8281 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcat
//
