LOCUS       sequence-272018-        3333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone contains nuclear EGFP 
            sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3333)
  AUTHORS   Sissaoui S, Yu J, Yan A, Li R, Zhu LJ, Kucukural A, Yukselen O, 
            Lawson ND
  TITLE     Genomic Characterization of Endothelial Enhancers Reveals a 
            Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene 
            Expression.
  JOURNAL   Circ Res. 2020 Feb 17. doi: 10.1161/CIRCRESAHA.119.316075.
  PUBMED    32065070
REFERENCE   2  (bases 1 to 3333)
  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: "Circ Res. 
            2020 Feb 17. doi: 10.1161/CIRCRESAHA.119.316075."
COMMENT     Sequence Label: pME-nEGFP
FEATURES             Location/Qualifiers
     source          1..3333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(272..299)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(391..477)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     526..548
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     540..557
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     541..557
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    573..672
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      675..684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(681..683,684..686,687..1397)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(681..683,684..686,687..1397)
                     /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(726..747)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(987..1006)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1334..1355
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1462..1561)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1578..1597)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1579..1597)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1602..1618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1602..1618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1720..1739)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1731..2540
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1798..1817)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2687..3275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3176..3195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
       61 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      121 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
      181 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta
      241 ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg
      301 cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg
      361 cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga
      421 caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg
      481 cctggcagtt ccctactctc gcgttaacgc tagcatggat gttttcccag tcacgacgtt
      541 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
      601 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      661 aaaaagcagg ctccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      721 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      781 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      841 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      901 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      961 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1021 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1081 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1141 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1201 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1261 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1321 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1381 tggacgagct gtacaagtcc ggactcagat ctcgacgagc tccaaagaag cgtaaggtag
     1441 atccaccgga tctagataac cacccagctt tcttgtacaa agttggcatt ataagaaagc
     1501 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1561 gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     1621 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     1681 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     1741 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     1801 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga
     1861 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     1921 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     1981 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag
     2041 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     2101 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     2161 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     2221 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     2281 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     2341 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     2401 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     2461 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     2521 tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta
     2581 cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt
     2641 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2701 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2761 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2821 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2881 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2941 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3001 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3061 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3121 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3181 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3241 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3301 ggttcctggc cttttgctgg ccttttgctc aca
//
