LOCUS       sequence-473487-        8336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A piggybac-based cloning vector containing mouse U6 promoter-driven 
            sgRNA for spCas9 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 8336)
  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 8336)
  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: "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
FEATURES             Location/Qualifiers
     source          1..8336
                     /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        1953..2256
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2535..3551
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3475..3497
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3559..3578
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3616..3636
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3640..3660
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3664..3684
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3700..3702,3703..3705,3706..4416)
                     /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(3745..3766)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4353..4374
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4417..4470
                     /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    4906..5027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4943..4962)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4997..5016
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5790..5824
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6315..6331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6315..6331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6328..6350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6339..6355
                     /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(6363..6369,6370..6387,6388..6393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6408..6429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6546..6563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6717..7305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6797..6816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7476..8267,8268..8336))
                     /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     8099..8118
                     /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 ccttgttggg gagacggacg tctccgttta agagctatgc tggaaacagc
     1741 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     1801 ctttttttct cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca
     1861 tgcatcccgg gtgactgact gagcggccgc actagttatt aatagtaatc aattacgggg
     1921 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1981 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     2041 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     2101 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     2161 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     2221 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     2281 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     2341 ttattttgtg cagcgatggg ggcggggggg ggggggggcg cgcgccrggc ggggsggggs
     2401 ggggsggggg gsggggsggg gsgrggcggr gaggkgcggc ggcagccaat cagagcggcg
     2461 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     2521 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     2581 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     2641 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
     2701 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
     2761 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
     2821 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     2881 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     2941 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     3001 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     3061 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     3121 tgccgggcgg ggcggggccg cctcgggccg gggrgggctc gggggagggg cgcggcggcc
     3181 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     3241 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     3301 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     3361 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     3421 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     3481 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     3541 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     3601 aattccgcca ccatgccaaa aaagaagaga aaggtagatc caaaaaagaa gagaaaggta
     3661 gatccaaaaa agaagagaaa ggtacacgtg tcaacgcgta tggtgagcaa gggcgaggag
     3721 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3781 ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc
     3841 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     3901 ggcgtggcct gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3961 gccatgcccg aaggctacgt ccaggagcgc accatctctt tcaaggacga cggtacctac
     4021 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4081 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac
     4141 agccacaacg tctatatcac ggccgacaag cagaagaacg gcatcaaggc taacttcaag
     4201 atccgccaca acgttgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4261 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca tcagtccgcc
     4321 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4381 gccgggatta cacatggcat ggacgagctg tacaaggagg gcagaggaag tcttctaaca
     4441 tgcggtgacg tggaggagaa tcccggccct atgaagacct tcaacatctc ccagcaggat
     4501 ctagaattag tagaagtagc gacagagaag attacaatgc tttatgagga taataaacat
     4561 catgtgggag cggcaattcg tacgaaaaca ggagaaatca tttcggcagt acatattgaa
     4621 gcgtatatag gacgagtaac tgtttgtgca gaagccattg cgattggtag tgcagtttcg
     4681 aatggacaaa aggattttga cacgattgta gctgttagac acccttattc tgacgaagta
     4741 gatagaagta ttcgagtggt aagtccttgt ggtatgtgta gggagttgat ttcagactat
     4801 gcaccagatt gttttgtgtt aatagaaatg aatggcaagt tagtcaaaac tacgattgaa
     4861 gaactcattc cactcaaata tacccgaaat taagttaact aactaaactt gtttattgca
     4921 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4981 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggaat
     5041 tgactcaaat gatgtcaatt agtctatcag aagctcatct ggtctccctt ccgggggaca
     5101 agacatccct gtttaatatt taaacagcag tgttcccaaa ctgggttctt atatcccttg
     5161 ctctggtcaa ccaggttgca gggtttcctg tcctcacagg aacgaagtcc ctaaagaaac
     5221 agtggcagcc aggtttagcc ccggaattga ctggattcct tttttagggc ccattggtat
     5281 ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca
     5341 gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct
     5401 cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc
     5461 tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccctgatat
     5521 ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat
     5581 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg
     5641 actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc
     5701 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt
     5761 tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct
     5821 agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc tcagtcatcg
     5881 cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt cccgcgaggt
     5941 tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg ttgagcgaaa
     6001 acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta acggtgaact
     6061 gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac agttccggat
     6121 ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga actgccgtgc
     6181 cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg aggacgggta
     6241 tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg gcgggcgcgc
     6301 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6361 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6421 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6481 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     6541 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6601 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6661 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6721 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6781 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6841 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6901 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6961 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     7021 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     7081 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     7141 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     7201 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     7261 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     7321 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     7381 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7441 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7501 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7561 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7621 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7681 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7741 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7801 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7861 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7921 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7981 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     8041 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     8101 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     8161 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     8221 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     8281 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcat
//
