LOCUS       sequence-248359-        7433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac transposon plasmid with CAG promoter GFP-Galectin 8 
            (GFP-Gal8) fusion protein. Useful as genetically encoded endosomal 
            escape sensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7433)
  AUTHORS   Rui Y, Wilson DR, Choi J, Varanasi M, Sanders K, Karlsson J, Lim M, 
            Green JJ
  TITLE     Carboxylated branched poly(beta-amino ester) nanoparticles enable 
            robust cytosolic protein delivery and CRISPR-Cas9 gene editing.
  JOURNAL   Sci Adv. 2019 Dec 6;5(12):eaay3255. doi: 10.1126/sciadv.aay3255. 
            eCollection 2019 Dec.
  PUBMED    31840076
REFERENCE   2  (bases 1 to 7433)
  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: "2019-12-6"; volume: "5(12):eaay3255. doi"; pages: " 
            10.1126/sciadv.aay3255. eCollection 2019 Dec"
COMMENT     Sequence Label: PB-GFP-Gal8
FEATURES             Location/Qualifiers
     source          1..7433
                     /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        748..1024
                     /label=chicken beta-actin promoter
     intron          1025..2041
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1965..1987
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2049..2068
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2134..2143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2140..2142,2143..2145,2146..2856)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2140..2142,2143..2145,2146..2856)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2185..2206)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2446..2465)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2793..2814
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4031..4050)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4096..4151
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4150..4169)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   4822..4856
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4879..4895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4879..4895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4892..4914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4903..4919
                     /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(4927..4933,4934..4951,4952..4957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4972..4993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5052..5247
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5098..5233
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5253..5387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5290..5309)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5344..5363
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5455..5472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5626..6214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5706..5725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6385..7176,7177..7245))
                     /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     7008..7027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7246..7350)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   join(7388..7433,1..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 agtatttacg gtaaactgcc cacttggcag
      601 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      661 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      721 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc
      781 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
      841 cagcgatggg ggcggggggg gggggggggc gcgcncaggc ggggcggggc ggggcgaggg
      901 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      961 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     1021 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     1081 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     1141 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
     1201 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
     1261 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
     1321 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1381 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1441 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1501 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1561 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1621 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1681 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1741 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1801 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1861 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1921 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1981 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     2041 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattctgcag
     2101 tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     2161 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2221 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2281 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2341 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2401 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2461 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2521 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2581 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2641 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2701 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2761 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2821 gccgggatca ctctcggcat ggacgagctg tacaaggaca tgtccatgat gttgtcctta
     2881 aacaacctac agaatatcat ctataacccg gtaatcccgt ttgttggcac cattcctgat
     2941 cagctggatc ctggaacttt gattgtgata cgtgggcatg ttcctagtga cgcagacaga
     3001 ttccaggtgg atctgcagaa tggcagcagc atgaaacctc gagccgatgt ggcctttcat
     3061 ttcaatcctc gtttcaaaag ggccggctgc attgtttgca atactttgat aaatgaaaaa
     3121 tggggacggg aagagatcac ctatgacacg cctttcaaaa gagaaaagtc ttttgagatc
     3181 gtgattatgg tgctgaagga caaattccag gtggctgtaa atggaaaaca tactctgctc
     3241 tatggccaca ggatcggccc agagaaaata gacactctgg gcatttatgg caaagtgaat
     3301 attcactcaa ttggttttag cttcagctcg gacttacaaa gtacccaagc atctagtctg
     3361 gaactgacag agataagtag agaaaatgtt ccaaagtctg gcacgcccca gcttcctagt
     3421 aatagaggag gagacatttc taaaatcgca cccagaactg tctacaccaa gagcaaagat
     3481 tcgactgtca atcacacttt gacttgcacc aaaataccac ctatgaacta tgtgtcaaag
     3541 aggctgccat tcgctgcaag gttgaacacc cccatgggcc ctggacgaac tgtcgtcgtt
     3601 aaaggagaag tgaatgcaaa tgccaaaagc tttaatgttg acctactagc aggaaaatca
     3661 aaggatattg ctctacactt gaacccacgc ctgaatatta aagcatttgt aagaaattct
     3721 tttcttcagg agtcctgggg agaagaagag agaaatatta cctctttccc atttagtcct
     3781 gggatgtact ttgagatgat aatttattgt gatgttagag aattcaaggt tgcagtaaat
     3841 ggcgtacaca gcctggagta caaacacaga tttaaagagc tcagcagtat tgacacgctg
     3901 gaaattaatg gagacatcca cttactggaa gtaaggagct ggtaggcggc cgcactcctc
     3961 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     4021 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     4081 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     4141 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     4201 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     4261 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4321 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4381 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4441 tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag agctccagcc
     4501 tacacaatcg ctcaagacgt gtaatgcttt tattatatat tagtcacgat atctataaca
     4561 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat
     4621 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc
     4681 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc
     4741 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga
     4801 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa
     4861 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4921 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4981 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5041 ccagcggatc cgcatctcaa taagtcagca accatagtcc cgcccctaac tccgcccatc
     5101 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     5161 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     5221 ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt
     5281 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5341 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg
     5401 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5461 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5521 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5581 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5641 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5821 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5941 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6001 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6061 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6121 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7261 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7321 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgggtc
     7381 gacttaaccc tagaaagata atcatattgt gacgtacgtt aaagataatc atg
//
