LOCUS       sequence-218274-        6466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac transposon plasmid for destabilized eGFP (GFPd2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6466)
  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 6466)
  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..6466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    314..347
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        443..746
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        749..1024
                     /label=chicken beta-actin promoter
     intron          1025..2033
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1957..1979
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2041..2060
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2126..2135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2132..2134,2135..2137,2138..2848,2849..2854,
                     2855..2977)
                     /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(2177..2198)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2438..2457)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2785..2806
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3067..3086)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3132..3187
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3186..3205)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   3855..3889
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3912..3928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3912..3928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3925..3947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3936..3952
                     /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(3960..3966,3967..3984,3985..3990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4005..4026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4085..4280
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4131..4266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4286..4420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4323..4342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4377..4396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4488..4505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4659..5247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4739..4758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5418..6209,6210..6278))
                     /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     6041..6060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6279..6383)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   join(6421..6466,1..17)
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 cgtaaaattg acgcatgtgt tttatcgatc tgtatatcga ggtttattta ttaatttgaa
       61 tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt
      121 tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta
      181 acaaaacttt taaacattct ctcttttaca aaaataaact tattttgtac tttaaaaaca
      241 gtcatgttgt attataaaat aagtaattag cttaacttat acataataga aacaaattat
      301 acttattaat cgcataactt cgtataatgt atgctatacg aagttatgcg attaagggat
      361 ctgtagggcg cagtagtcca actagttatt aatagtaatc aattacgggg tcattagttc
      421 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      541 tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag
      601 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      661 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      721 acgtattagt catcgctatt accatgggtc gaggtgagcc ccacgttctg cttcactctc
      781 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      841 gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      901 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      961 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     1021 ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc
     1081 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1141 tccgggctgt aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga
     1201 aagccttaaa gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt
     1261 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg
     1321 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg
     1381 ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1441 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
     1501 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tgcggggcgt
     1561 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1621 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggccccgg agcgccggcg
     1681 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1741 agggacttcc tttgtcccaa atctggcgga gccgaaatct gggaggcgcc gccgcacccc
     1801 ctctagcggg cgcgggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1861 cttcgtgcgt cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg
     1921 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1981 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     2041 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattctgc agtcgacggt
     2101 accgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac
     2161 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2221 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2281 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     2341 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2401 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2461 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2521 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2581 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2641 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2701 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2761 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2821 cactctcggc atggacgagc tgtacaagaa gcttagccat ggcttcccgc cggaggtgga
     2881 ggagcaggat gatggcacgc tgcccatgtc ttgtgcccag gagagcggga tggaccgtca
     2941 ccctgcagcc tgtgcttctg ctaggatcaa tgtgtagatg cgcggccgca ctcctcaggt
     3001 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     3061 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     3121 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     3181 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     3241 ggtttagagt ttggcaacat atgccatatg ctggctgcca tgaacaaagg tggctataaa
     3301 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     3361 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     3421 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     3481 tcatagctgt ccctcttctc ttatgaagat ccctcgacct gcaggctcca gcctacacaa
     3541 tcgctcaaga cgtgtaatgc ttttattata tattagtcac gatatctata acaagaaaat
     3601 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga
     3661 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt
     3721 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc
     3781 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca
     3841 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taaaagcttg
     3901 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3961 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4021 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4081 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4141 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4201 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4261 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     4321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4381 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     4441 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4501 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4561 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4621 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4681 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4741 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4801 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     4861 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4921 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4981 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5041 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5101 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5161 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5221 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5281 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5341 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5401 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5461 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5521 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5581 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5641 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5701 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5761 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5821 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5881 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5941 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6001 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6061 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6121 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6181 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6241 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6301 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6361 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtcgacttaa
     6421 ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatg
//
