LOCUS       sequence-209373-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transposon donor plasmid for long-term expression of membrane 
            targetted GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  AUTHORS   Lancaster MA, Corsini NS, Wolfinger S, Gustafson EH, Phillips AW, 
            Burkard TR, Otani T, Livesey FJ, Knoblich JA
  TITLE     Guided self-organization and cortical plate formation in human brain
            organoids.
  JOURNAL   Nat Biotechnol. 2017 Jul;35(7):659-666. doi: 10.1038/nbt.3906. Epub 
            2017 May 31.
  PUBMED    28562594
REFERENCE   2  (bases 1 to 6352)
  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"; doi: "10.1038/nbt.3906"; 
            journalName: "Nat Biotechnol"; date: "2017-07"; volume: "35"; issue:
            "7"; pages: "659-666"
COMMENT     Sequence Label: pT2-CAG-fGFP
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..581
                     /label=chicken beta-actin promoter
     intron          582..1598
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1522..1544
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1606..1625
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1740..1757
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    1767..1791
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1803..1812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1809..1811,1812..1814,1815..2525)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1809..1811,1812..1814,1815..2525)
                     /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(1854..1875)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2115..2134)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2462..2483
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2684..2708)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3123..3143)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3123..3141)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3162..3178)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3162..3178)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3175..3197)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3186..3202
                     /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(3210..3216,3217..3234,3235..3240))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3255..3276
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3393..3410)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3564..4152)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3644..3663)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4323..5114,5115..5183))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4946..4965
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5184..5288)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5315..5770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5402..5421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5612..5633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5896..5918
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5910..5927
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5911..5927
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5937..5956
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5937..5955
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggggcgcgc gccaggcggg gcgggggggg gggggggggg ggggggggcg aggcggagag
      481 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      541 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      601 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      661 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      721 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      781 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      841 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      901 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      961 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1021 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1081 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1141 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1201 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1261 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1321 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1381 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1441 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1501 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1561 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1621 ttgtgctgtc tcatcatttt ggcaaagaat tctgcagtcg acggtaccgc gggcccggga
     1681 tccaccggtc actgcagtta attaaggccg gcccgggcgc gcctaggacc tcgtactacc
     1741 atcaccatca ccatcacctc gaatcaacaa gtttgtacaa aaaagcaggc tcgctaccgg
     1801 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1861 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1921 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1981 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2041 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2101 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2161 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2221 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2281 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2341 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2401 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2461 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2521 acaagtccgg actcagatct cgagctcaag ctgcacgcgt gagtaagctg aaccctcctg
     2581 atgagagtgg ccccggctgc atgagctgca agtgtgtgct ctcctgaaat tctgcagtcg
     2641 acggtaccgc gggcccggga tccaccggat ctagataact gatacccagc tttcttgtac
     2701 aaagtggttg attccaatgc atgcggggac tagtgcgcgc ggccgctcta gatggccaga
     2761 tctagcttgt ggaaggctac tcgaaatgtt tgacccaagt taaacaattt aaaggcaatg
     2821 ctaccaaata ctaattgagt gtatgtaaac ttctgaccca ctgggaatgt gatgaaagaa
     2881 ataaaagctg aaatgaatca ttctctctac tattattctg atatttcaca ttcttaaaat
     2941 aaagtggtga tcctaactga cctaagacag ggaattttta ctaggattaa atgtcaggaa
     3001 ttgtgaaaaa gtgagtttaa atgtatttgg ctaaggtgta tgtaaacttc cgacttcaac
     3061 tgtataggga tcctctagct agagtcgacc tcgagggggg gcccggtacc cagcttttgt
     3121 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     3181 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3241 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3301 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3361 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3421 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3481 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3541 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3601 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3661 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3721 tccgcctttc tcccttcggg aaccgtggcg ctttctcata gctcacgctg taggtatctc
     3781 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3841 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3901 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3961 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4021 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4081 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4141 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4201 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4261 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4321 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4381 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4441 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4501 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4561 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4621 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4681 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4741 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta acttggccgc agtgttatca
     4801 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4861 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4921 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4981 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5041 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5101 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5161 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5221 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5281 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
     5341 gcggcgggag tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5401 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5461 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5521 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5581 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5641 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5701 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5761 cttacaattt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     5821 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     5881 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     5941 acgactcact atagggcgaa ttggagctcg gatccctata cagttgaagt cggaagttta
     6001 catacactta agttggagtc attaaaactc gtttttcaac tactccacaa atttcttgtt
     6061 aacaaacaat agttttggca agtcagttag gacatctact ttgtgcatga cacaagtcat
     6121 ttttccaaca attgtttaca gacagattat ttcacttata attcactgta tcacaattcc
     6181 agtgggtcag aagtttacat acactaagtt gactgtgcct ttaaacagct tggaaaattc
     6241 cagaaaatga tgtcatggct ttagaagctt cctgacctgg tgcgcattag tctagttatt
     6301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cg
//
