LOCUS       sequence-306427-        7943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac transposon vector with CAG promoter expressing Otx2 and 
            EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7943)
  AUTHORS   Ballabio C, Anderle M, Gianesello M, Lago C, Miele E, Cardano M, 
            Aiello G, Piazza S, Caron D, Gianno F, Ciolfi A, Pedace L, 
            Mastronuzzi A, Tartaglia M, Locatelli F, Ferretti E, Giangaspero F, 
            Tiberi L
  TITLE     Modeling medulloblastoma in vivo and with human cerebellar 
            organoids.
  JOURNAL   Nat Commun. 2020 Jan 29;11(1):583. doi: 10.1038/s41467-019-13989-3.
  PUBMED    31996670
REFERENCE   2  (bases 1 to 7943)
  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: "2020-01-29"; volume: "11(1):583. doi"; pages: " 
            10.1038/s41467-019-13989-3"
COMMENT     Sequence Label: pPB CAG Otx2-IRES-EGFP
FEATURES             Location/Qualifiers
     source          1..7943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(96..887,888..956))
                     /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     719..738
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(957..1061)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1087..1542)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1224..1245)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1436..1455
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1669..1691
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1683..1700
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1684..1700
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1707..1726
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1707..1725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1744..1778
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2061..2440
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2442..2719
                     /label=chicken beta-actin promoter
     intron          2720..3736
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3660..3682
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3744..3763
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    join(4708..5282,5283..5285,5286..5294)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4875..4892)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5102..5121
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5295..5297,5298..5300,5301..6014)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5295..5297,5298..5300,5301..6011)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5340..5361)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5601..5620)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5948..5969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6106..6125)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6171..6226
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6225..6244)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   6754..6816
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(6840..6858)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6879..6895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6879..6895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6892..6914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6903..6919
                     /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(6927..6933,6934..6951,6952..6957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6972..6993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7110..7127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7281..7868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7360..7379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
       61 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      121 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      181 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
      241 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
      301 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      361 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      421 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      481 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      541 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      601 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      661 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      721 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      781 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      841 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      901 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      961 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1021 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1081 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1141 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     1201 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1261 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1321 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1381 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     1441 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     1501 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     1561 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     1621 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     1681 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtacct
     1741 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
     1801 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
     1861 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
     1921 cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga
     1981 taattatatt gttatttcat gttctactta cgtgataact tattatatat atattttctt
     2041 gttatagata tcgtcgggtc gacattgatt attgactagt tattaatagt aatcaattac
     2101 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     2161 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     2221 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     2281 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     2341 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2401 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
     2461 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
     2521 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc caggcggggc
     2581 ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
     2641 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
     2701 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
     2761 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     2821 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
     2881 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag
     2941 cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct
     3001 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg
     3061 cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa
     3121 aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg
     3181 gctgcaaccc cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc
     3241 ggggctccgt acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt
     3301 gggggtgccg ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg
     3361 cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg
     3421 gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc
     3481 tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag
     3541 gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct
     3601 ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg
     3661 gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt
     3721 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     3781 caaagaattg ctcgagatga tgtcttatct aaagcaaccg ccttacgcag tcaatgggct
     3841 gagtctgacc acttcgggta tggacttgct gcatccctcc gtgggctacc ccgccacccc
     3901 ccggaaacag cgaagggaga ggacgacttt tactagggca cagctcgacg ttctggaagc
     3961 tctgtttgcc aagacccggt acccagacat cttcatgagg gaagaggtgg cactgaaaat
     4021 caacttgcca gaatccaggg tgcaggtatg gtttaagaat cgaagagcta agtgccgcca
     4081 acagcagcag cagcagcaga atggaggtca gaacaaagtg aggcctgcca agaagaagag
     4141 ctctccagct cgggaagtga gttcagagag tggaacaagt ggccagttca ctcccccctc
     4201 tagtacctca gtcccaacca ttgccagcag cagtgcccca gtgtctatct ggagtccagc
     4261 gtccatctcc ccactgtctg accccttgtc cacttcctcc tcctgcatgc agaggtccta
     4321 tcccatgacc tatactcagg cttcaggtta tagtcaaggc tatgctggct caacttccta
     4381 ctttgggggc atggactgtg gatcttattt gacccctatg catcaccagc ttcctggacc
     4441 aggggccaca ctcagtccca tgggtaccaa tgctgttacc agccatctca atcagtcccc
     4501 agcttctctt tccacccagg gatatggagc ttcaagcttg ggttttaact caaccactga
     4561 ttgcttggat tataaggacc aaactgcctc ttggaagctt aacttcaatg ctgactgctt
     4621 ggattataaa gatcagacgt cctcatggaa attccaggtt ttgtgatgag aattctgcag
     4681 tcgacggtac cgcgggcccg ggatccgccc ctctccctcc ccccccccta acgttactgg
     4741 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     4801 gccgtctttt ggcaatgtga gggtccggaa acctggccct gtcttcttga cgagcattcc
     4861 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     4921 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     4981 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     5041 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     5101 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     5161 tatgggatct gatctggggc ctcggtacac atgctttaca tgtgtttagt cgaggttaaa
     5221 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     5281 atatggccac aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     5341 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     5401 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     5461 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     5521 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     5581 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     5641 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     5701 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     5761 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     5821 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     5881 tgcccgacaa ccactacctg agcactcagt ccgccctgag caaagacccc aacgagaagc
     5941 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     6001 agctgtacaa gtaaagcggc cgcaattcac tcctcaggtg caggctgcct atcagaaggt
     6061 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc
     6121 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     6181 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     6241 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     6301 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     6361 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     6421 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     6481 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     6541 cttatggaga tccctcgacc tgcaccgtct atggagatcg gatcctttgt tactttatag
     6601 aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt
     6661 tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa
     6721 taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc
     6781 tttaacgtac gtcacaatat gattatcttt ctagggttaa agagctccag cttttgttcc
     6841 ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga
     6901 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6961 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     7021 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     7081 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     7141 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     7201 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     7261 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     7321 cgacgctcaa gtcagaggtg gcgaacccga caggactata aagataccag gcgtttcccc
     7381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     7441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     7501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     7741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7921 cacgttaagg gattttggtc atg
//
