LOCUS       sequence-54310-e        6857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6857)
  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 6857)
  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..6857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   11..73
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    370..403
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        499..802
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        805..1080
                     /label=chicken beta-actin promoter
     primer_bind     2013..2035
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             join(2048..2050,2051..2053,2054..2764)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2048..2050,2051..2053,2054..2764)
                     /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(2093..2114)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2354..2373)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2701..2722
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2774..2827
                     /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             2834..3511
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2834..3511
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2964..2982)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3232..3251
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3426..3449
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3515..3534)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3580..3635
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3634..3653)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   4303..4337
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4360..4376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4360..4376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4373..4395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4384..4400
                     /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(4408..4414,4415..4432,4433..4438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4453..4474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4533..4728
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4579..4714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4734..4868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4771..4790)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4825..4844
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4936..4953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5107..5695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5187..5206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5866..6657,6658..6726))
                     /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     6489..6508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6727..6831)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta
       61 aaattgacgc atgtgtttta tcgatctgta tatcgaggtt tatttattaa tttgaataga
      121 tattaagttt tattatattt acacttacat actaataata aattcaacaa acaatttatt
      181 tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa
      241 aacttttaaa cattctctct tttacaaaaa taaacttatt ttgtacttta aaaacagtca
      301 tgttgtatta taaaataagt aattagctta acttatacat aatagaaaca aattatactt
      361 attaatcgca taacttcgta taatgtatgc tatacgaagt tatgcgatta agggatctgt
      421 agggcgcagt agtccaacta gttattaata gtaatcaatt acggggtcat tagttcatag
      481 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      541 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      601 gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact tggcagtaca
      661 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      721 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      781 attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc actctcccca
      841 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      901 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      961 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     1021 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1081 ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc
     1141 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
     1201 ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc
     1261 cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt
     1321 gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc
     1381 gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg gccgggggcg
     1441 gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     1501 gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc
     1561 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg gggcgtggcg
     1621 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
     1681 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg ccggcggctg
     1741 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     1801 acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg caccccctct
     1861 agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc
     1921 gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc cgcaggggga
     1981 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     2041 ctctagaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2101 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2161 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2221 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2281 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2341 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2401 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2461 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2521 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2581 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2641 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2701 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2761 caagggctcc ggcgagggca ggggaagtct actaacatgc ggggacgtgg aggaaaatcc
     2821 cggcccagaa ttcatggcct cctccgagga cgtcatcaag gagttcatgc gcttcaaggt
     2881 gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     2941 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
     3001 cgcctgggac atcctgtccc ctcagttcca gtacggctcc aaggcctacg tgaagcaccc
     3061 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     3121 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     3181 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     3241 gcagaagaag accatgggct gggaggcctc caccgagcgg atgtaccccg aggacggcgc
     3301 cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact acgacgccga
     3361 ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg cccggcgcct acaagaccga
     3421 catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg
     3481 cgccgagggc cgccactcca ccggcgccta aagatctttt tccctctgcc aaaaattatg
     3541 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     3601 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     3661 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gccatatgct
     3721 ggctgccatg aacaaaggtg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     3781 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     3841 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     3901 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atgaagatcc
     3961 ctcgacctgc aggctccagc ctacacaatc gctcaagacg tgtaatgctt ttattatata
     4021 ttagtcacga tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     4081 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     4141 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     4201 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     4261 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     4321 agactatctt tctagggtta aaagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4381 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4441 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4501 ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc
     4561 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4621 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4681 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt
     4741 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4801 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4861 tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5281 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5521 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5761 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5821 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5881 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5941 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6001 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6061 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6121 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6181 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6241 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6301 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6361 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6421 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6481 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6541 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6601 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6661 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6721 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6841 ccgaaaagtg ccacctg
//
