LOCUS       sequence-442830-        3546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning of GFP overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3546)
  AUTHORS   Pulice JL, Meyerson M
  TITLE     Dosage amplification dictates oncogenic regulation by the NKX2-1 
            lineage factor in lung adenocarcinoma.
  JOURNAL   bioRxiv [Preprint]. 2023 Oct 27:2023.10.26.563996. doi: 
            10.1101/2023.10.26.563996.
  PUBMED    37994369
REFERENCE   2  (bases 1 to 3546)
  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: "bioRxiv 
            [Preprint]. 2023 Oct 27:2023.10.26.563996. doi: 
            10.1101/2023.10.26.563996."
COMMENT     Sequence Label: pDONR223_GFP
FEATURES             Location/Qualifiers
     source          1..3546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(121..123,124..126,127..837)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(166..187)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(427..446)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     774..795
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(857..956)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(973..992)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(974..992)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(997..1013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(997..1013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1444..2235
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2328..2916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2817..2836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3070..3087
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3246..3273)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3365..3451)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3500..3522
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3514..3531
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3515..3531
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgcggccgc ccccttcacc
      121 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      181 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac
      241 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      301 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      361 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      421 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      481 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      541 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      601 ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      661 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      721 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      781 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
      841 aagggtgggc gcgccgaccc agctttcttg tacaaagttg gcattataag aaagcattgc
      901 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
      961 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     1021 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     1081 ctcctctaga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     1141 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     1201 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     1261 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     1321 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     1381 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     1441 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     1501 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     1561 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     1621 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     1681 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     1741 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     1801 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     1861 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     1921 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     1981 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     2041 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     2101 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     2161 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     2221 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     2281 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2341 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2401 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2461 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2521 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2581 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2641 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2701 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2761 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2821 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2881 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2941 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3001 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3061 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     3121 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3181 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     3241 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     3301 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     3361 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     3421 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     3481 acgctagcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc
     3541 gggccc
//
