LOCUS       sequence-223039-        6414 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Piggybac transposon plasmid for destabilized eGFP (GFPd2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6414)
  AUTHORS   Rui Y, Wilson D, Sanders K, Green JJ
  TITLE     Reducible branched ester-amine quadpolymers (rBEAQs) co-delivering 
            plasmid DNA and RNA oligonucleotides enable CRISPR/Cas9 genome 
            editing.
  JOURNAL   ACS Appl Mater Interfaces. 2019 Feb 22. doi: 10.1021/acsami.8b20206.
  PUBMED    30794383
REFERENCE   2  (bases 1 to 6414)
  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: "ACS Appl 
            Mater Interfaces. 2019 Feb 22. doi: 10.1021/acsami.8b20206."
COMMENT     Sequence Label: PB-CAG-GFPd2
FEATURES             Location/Qualifiers
     source          1..6414
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          108..1124
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1048..1070
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1132..1151
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1217..1226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1223..1225,1226..1228,1229..1939,1940..1945,
                     1946..2068)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(1268..1289)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1529..1548)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1876..1897
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2154..2173)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2219..2274
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2273..2292)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2945..2979
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3002..3018)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3002..3018)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3015..3037)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3026..3042
                     /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(3050..3056,3057..3074,3075..3080))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3095..3116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3175..3370
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3221..3356
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3376..3510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3413..3432)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3467..3486
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3578..3595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3749..4337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3829..3848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4508..5299,5300..5368))
                     /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     5131..5150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5369..5473)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   5511..5573
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5870..5903
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        5999..6302
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
       61 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
      121 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
      181 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
      241 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
      301 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
      361 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
      421 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
      481 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
      541 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
      601 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
      661 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
      721 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
      781 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
      841 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
      901 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
      961 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     1021 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     1081 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     1141 tggttattgt gctgtctcat cattttggca aagaattctg cagtcgacgg taccgcgggc
     1201 ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     1261 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1321 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1381 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1441 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1501 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1561 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1621 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1681 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1741 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1801 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1861 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1921 catggacgag ctgtacaaga agcttagcca tggcttcccg ccggaggtgg aggagcagga
     1981 tgatggcacg ctgcccatgt cttgtgccca ggagagcggg atggaccgtc accctgcagc
     2041 ctgtgcttct gctaggatca atgtgtaggc ggccgcactc ctcaggtgca ggctgcctat
     2101 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt
     2161 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     2221 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     2281 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     2341 gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag
     2401 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt
     2461 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc
     2521 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc
     2581 cctcttctct tatggagatc cctcgacctg cagagctcca gcctacacaa tcgctcaaga
     2641 cgtgtaatgc ttttattata tattagtcac gatatctata acaagaaaat atatatataa
     2701 taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt
     2761 aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa
     2821 atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag
     2881 atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag
     2941 aatgcatgcg tcaattttac gcagactatc tttctagggt taaaagcttg gcgtaatcat
     3001 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3061 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3121 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct
     3181 caataagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3241 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3301 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3361 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3421 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3481 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     3541 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3601 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3661 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3721 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3781 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3841 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3901 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3961 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4021 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4081 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4141 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4201 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4261 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4321 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4381 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4441 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4501 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4561 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4621 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4681 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4741 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4801 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4861 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4921 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4981 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5041 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5101 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5161 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5221 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5281 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5341 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5401 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5461 taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtcgacttaa ccctagaaag
     5521 ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta
     5581 tcgatctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt
     5641 acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa
     5701 aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa cattctctct
     5761 tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta taaaataagt
     5821 aattagctta acttatacat aatagaaaca aattatactt attaatcgca taacttcgta
     5881 taatgtatgc tatacgaagt tatgcgatta agggatctgt agggcgcagt agtccaacta
     5941 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     6001 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     6061 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     6121 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     6181 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     6241 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6301 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     6361 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggg
//
