LOCUS       sequence-204980-        6328 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 6328)
  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 6328)
  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..6328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..56
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     48..65
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     49..65
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     75..94
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        75..93
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        491..794
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        797..1072
                     /label=chicken beta-actin promoter
     intron          1073..2085
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2009..2031
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2093..2112
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2227..2244
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    2254..2278
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2284..2286,2287..2289,2290..3000)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2284..2286,2287..2289,2290..3000)
                     /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(2329..2350)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2590..2609)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2937..2958
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3150..3174)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3589..3609)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3589..3607)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3628..3644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3628..3644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3641..3663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3652..3668
                     /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(3676..3682,3683..3700,3701..3706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3721..3742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3859..3876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4030..4618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4110..4129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4789..5580,5581..5649))
                     /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     5412..5431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5650..5754)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5781..6236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5868..5887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6078..6099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
       61 ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccctataca
      121 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta
      181 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt
      241 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat
      301 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt
      361 aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttcc tgacctggtg
      421 cgcattagtc tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      481 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      541 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      601 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      661 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      721 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      781 tcgctattac catgggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
      841 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
      901 gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      961 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     1021 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     1081 tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg
     1141 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     1201 ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg
     1261 gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt
     1321 gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     1381 cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg
     1441 cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     1501 gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga
     1561 gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc
     1621 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
     1681 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
     1741 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
     1801 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
     1861 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     1921 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     1981 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     2041 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
     2101 ctggttattg tgctgtctca tcattttggc aaagaattct gcagtcgacg gtaccgcggg
     2161 cccgggatcc accggtcact gcagttaatt aaggccggcc cgggcgcgcc taggacctcg
     2221 tactaccatc accatcacca tcacctcgaa tcaacaagtt tgtacaaaaa agcaggctcc
     2281 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2341 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2401 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2461 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2521 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2581 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2641 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2701 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2761 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2821 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2881 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2941 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3001 tccggactca gatctcgagc tcaagctgca cgcgtgagta agctgaaccc tcctgatgag
     3061 agtggccccg gctgcatgag ctgcaagtgt gtgctctcct gaaattctgc agtcgacggt
     3121 accgcgggcc cgggatccac cggatctaga cccagctttc ttgtacaaag tggttgattc
     3181 caatgcatgc ggggactagt gcgcgcggcc gctctagatg gccagatcta gcttgtggaa
     3241 ggctactcga aatgtttgac ccaagttaaa caatttaaag gcaatgctac caaatactaa
     3301 ttgagtgtat gtaaacttct gacccactgg gaatgtgatg aaagaaataa aagctgaaat
     3361 gaatcattct ctctactatt attctgatat ttcacattct taaaataaag tggtgatcct
     3421 aactgaccta agacagggaa tttttactag gattaaatgt caggaattgt gaaaaagtga
     3481 gtttaaatgt atttggctaa ggtgtatgta aacttccgac ttcaactgta tagggatcct
     3541 ctagctagag tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg
     3601 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3661 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3721 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3781 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3841 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3901 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3961 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4021 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4081 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4141 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4201 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4261 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4321 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4381 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4441 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4501 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4561 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4621 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4681 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4741 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4801 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4861 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4921 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4981 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5041 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5101 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5161 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5221 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5281 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5341 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5401 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5461 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5521 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5581 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5641 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5701 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5761 tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5821 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5881 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5941 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     6001 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6061 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     6121 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     6181 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat
     6241 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     6301 cgccagctgg cgaaaggggg atgtgctg
//
