LOCUS       sequence-267264-        3331 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 3331)
  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 3331)
  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..3331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(677..679,680..682,683..1393)
                     /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(722..743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(983..1002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1330..1351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1458..1557)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1574..1593)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1575..1593)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1598..1614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1598..1614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1716..1735)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1727..2536
                     /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(1794..1813)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2683..3271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3172..3191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1381 cgagctgtac aagtccggac tcagatctcg acgagctcca aagaagcgta aggtagatcc
     1441 accggatcta gataaccacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     1501 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     1561 tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     1621 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaataa tatcatcatg
     1681 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1741 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     1801 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     1861 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     1921 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     1981 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     2041 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     2101 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     2161 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     2221 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     2281 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     2341 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     2401 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     2461 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     2521 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     2581 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     2641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2821 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3301 cctggccttt tgctggcctt ttgctcacat g
//
