LOCUS       sequence-177247-        5742 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5742)
  AUTHORS   Chen F, LoTurco J
  TITLE     A method for stable transgenesis of radial glia lineage in rat 
            neocortex by piggyBac mediated transposition.
  JOURNAL   J Neurosci Methods. 2012 Jun 15;207(2):172-80. Epub 2012 Apr 11.
  PUBMED    22521325
REFERENCE   2  (bases 1 to 5742)
  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: "J Neurosci 
            Methods."; date: "2012-06-15"; volume: "207(2)"; pages: "172-80. 
            Epub 2012 Apr 11"
COMMENT     Sequence Label: PBCAG-eGFPt2amRFP
FEATURES             Location/Qualifiers
     source          1..5742
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     39..61
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             join(74..76,77..79,80..790)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(74..76,77..79,80..790)
                     /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(119..140)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(380..399)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     727..748
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             800..853
                     /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"
     CDS             860..1537
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRPS
                     TGA"
     primer_bind     complement(990..1008)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1258..1277
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1452..1475
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1541..1560)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1606..1661
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1660..1679)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2332..2366
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2389..2405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2389..2405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2402..2424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2413..2429
                     /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(2437..2443,2444..2461,2462..2467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2482..2503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2562..2757
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2608..2743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2763..2897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2800..2819)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2854..2873
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2965..2982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3136..3724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3216..3235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3895..4686,4687..4755))
                     /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     4518..4537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4756..4860)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   4898..4960
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5257..5290
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        5386..5689
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
       61 cggcggctct agaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      121 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      181 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      241 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
      301 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
      361 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
      421 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
      481 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
      541 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
      601 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
      661 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
      721 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
      781 gctgtacaag ggctccggcg agggcagggg aagtctacta acatgcgggg acgtggagga
      841 aaatcccggc ccagaattca tggcctcctc cgaggacgtc atcaaggagt tcatgcgctt
      901 caaggtgcgc atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      961 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct
     1021 gcccttcgcc tgggacatcc tgtcccctca gttccagtac ggctccaagg cctacgtgaa
     1081 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     1141 gcgcgtgatg aacttcgagg atggcggcgt ggtgaccgtg acccaggact cctccctgca
     1201 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     1261 cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcggatgt accccgagga
     1321 cggcgccctg aagggcgaga tcaagatgag gctgaagctg aaggacggcg gccactacga
     1381 cgccgaggtc aagaccacct acatggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1441 gaccgacatc aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1501 cgagcgcgcc gagggccgcc cctccaccgg cgcctaaaga tctttttccc tctgccaaaa
     1561 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     1621 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     1681 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     1741 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     1801 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     1861 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     1921 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     1981 ggagatccct cgacctgcag agctccagcc tacacaatcg ctcaagacgt gtaatgcttt
     2041 tattatatat tagtcacgat atctataaca agaaaatata tatataataa gttatcacgt
     2101 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     2161 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     2221 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     2281 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     2341 attttacgca gactatcttt ctagggttaa aagcttggcg taatcatggt catagctgtt
     2401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2521 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa taagtcagca
     2581 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2641 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2701 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2761 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2821 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2881 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2941 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3001 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3061 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3121 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3181 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3241 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3301 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3361 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3421 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3481 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3541 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3601 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3661 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3721 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3781 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3841 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3901 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3961 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4021 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4081 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4141 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4201 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4261 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4321 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4381 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4441 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4501 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4561 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4621 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4681 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4741 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4801 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4861 cacatttccc cgaaaagtgc cacctgggtc gacttaaccc tagaaagata atcatattgt
     4921 gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg atctgtatat
     4981 cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca cttacatact
     5041 aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa aaaacaaaaa
     5101 ctcaaaattt cttctataaa gtaacaaaac ttttaaacat tctctctttt acaaaaataa
     5161 acttattttg tactttaaaa acagtcatgt tgtattataa aataagtaat tagcttaact
     5221 tatacataat agaaacaaat tatacttatt aatcgcataa cttcgtataa tgtatgctat
     5281 acgaagttat gcgattaagg gatctgtagg gcgcagtagt ccaactagtt attaatagta
     5341 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     5401 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     5461 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     5521 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     5581 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     5641 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
     5701 ccccacgttc tgcttcactc tccccatctc ccccccctcc cc
//
