LOCUS       sequence-477909-        8339 bp    DNA     circular SYN 08-JUL-2025
DEFINITION  A piggybac-based vector containing mouse U6 promoter-driven EIF3D 
            sgRNA #1 and CAG promoter-driven nuclear-localized Clover-P2A-BSR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8339)
  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 8339)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 8, 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_sgEIF3D-1
FEATURES             Location/Qualifiers
     source          1..8339
                     /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"
     intron          2538..3554
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3478..3500
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3562..3581
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3619..3639
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3643..3663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3667..3687
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3703..3705,3706..3708,3709..4419)
                     /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(3748..3769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4356..4377
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4420..4473
                     /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    4909..5030
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4946..4965)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5000..5019
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5793..5827
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6318..6334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6318..6334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6331..6353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6342..6358
                     /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(6366..6372,6373..6390,6391..6396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6411..6432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6549..6566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6720..7308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6800..6819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7479..8270,8271..8339))
                     /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     8102..8121
                     /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 ccttgttgga cgcgggacac gaccttgtgt 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 gcgcgcgcca ggsggggsgg
     2401 gggggggggg gggggggggg ggggsgrggs ggrgrggkgs ggcggcagcc aatcagagcg
     2461 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     2521 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     2581 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     2641 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     2701 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     2761 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     2821 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     2881 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2941 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     3001 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     3061 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     3121 gggtgccggg cggggcgggg ccgcckcggg ccggggrggg ctcgggggrg gggcgcggcg
     3181 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     3241 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     3301 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     3361 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     3421 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     3481 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     3541 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     3601 aagaattccg ccaccatgcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag
     3661 gtagatccaa aaaagaagag aaaggtacac gtgtcaacgc gtatggtgag caagggcgag
     3721 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3781 aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
     3841 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
     3901 tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     3961 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc
     4021 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     4081 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc
     4141 aacagccaca acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc
     4201 aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     4261 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc
     4321 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     4381 gccgccggga ttacacatgg catggacgag ctgtacaagg agggcagagg aagtcttcta
     4441 acatgcggtg acgtggagga gaatcccggc cctatgaaga ccttcaacat ctcccagcag
     4501 gatctagaat tagtagaagt agcgacagag aagattacaa tgctttatga ggataataaa
     4561 catcatgtgg gagcggcaat tcgtacgaaa acaggagaaa tcatttcggc agtacatatt
     4621 gaagcgtata taggacgagt aactgtttgt gcagaagcca ttgcgattgg tagtgcagtt
     4681 tcgaatggac aaaaggattt tgacacgatt gtagctgtta gacaccctta ttctgacgaa
     4741 gtagatagaa gtattcgagt ggtaagtcct tgtggtatgt gtagggagtt gatttcagac
     4801 tatgcaccag attgttttgt gttaatagaa atgaatggca agttagtcaa aactacgatt
     4861 gaagaactca ttccactcaa atatacccga aattaagtta actaactaaa cttgtttatt
     4921 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4981 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     5041 aattgactca aatgatgtca attagtctat cagaagctca tctggtctcc cttccggggg
     5101 acaagacatc cctgtttaat atttaaacag cagtgttccc aaactgggtt cttatatccc
     5161 ttgctctggt caaccaggtt gcagggtttc ctgtcctcac aggaacgaag tccctaaaga
     5221 aacagtggca gccaggttta gccccggaat tgactggatt ccttttttag ggcccattgg
     5281 tatggctttt tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt
     5341 tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg
     5401 gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc
     5461 ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctga
     5521 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     5581 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     5641 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     5701 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     5761 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     5821 tctagggtta atctagctgc atcaggatca tatcgtcggg tcttttttcc ggctcagtca
     5881 tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga
     5941 ggttgaagcg gcatggaaag agtttgccga ggatgactgc tgctgcattg acgttgagcg
     6001 aaaacgcacg tttaccatga tgattcggga aggtgtggcc atgcacgcct ttaacggtga
     6061 actgttcgtt caggccacct gggataccag ttcgtcgcgg cttttccgga cacagttccg
     6121 gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg
     6181 tgccggtgtg cagattaatg acagcggtgc ggcgctggga tattacgtca gcgaggacgg
     6241 gtatcctggc tggatgccgc agaaatggac atggataccc cgtgagttac ccggcgggcg
     6301 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6361 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6421 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6481 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7141 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7441 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7501 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7561 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7621 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7681 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7741 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7801 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7861 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7921 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7981 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8041 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8101 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8161 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8221 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8281 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcat
//
