LOCUS       sequence-178533-        3829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway Mid-entry clone containing promoter sequence specific for 
            cldn5b driving expression of eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3829)
  AUTHORS   Quillien A, Abdalla M, Yu J, Ou J, Zhu LJ, Lawson ND
  TITLE     Robust Identification of Developmentally Active Endothelial 
            Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq.
  JOURNAL   Cell Rep. 2017 Jul 18;20(3):709-720. doi: 
            10.1016/j.celrep.2017.06.070.
  PUBMED    28723572
REFERENCE   2  (bases 1 to 3829)
  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: 
            "10.1016/j.celrep.2017.06"; journalName: "Cell Rep"; date: 
            "2017-07-18- 18"; volume: "20"; issue: "3"; pages: "709-720"
COMMENT     Sequence Label: pMEcldn5bP1gfp
FEATURES             Location/Qualifiers
     source          1..3829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     492..511
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     745..762
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(921..948)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1040..1126)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1175..1197
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1189..1206
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1190..1206
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1222..1321
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1798..1807
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1804..1806,1807..1809,1810..2520)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1804..1806,1807..1809,1810..2520)
                     /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(1849..1870)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2110..2129)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2457..2478
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2521..2586
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(2607..2706)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2723..2742)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2724..2742)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2747..2763)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2747..2763)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2865..2884)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2876..3685
                     /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(2943..2962)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
       61 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      121 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
      181 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      241 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      301 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      361 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      421 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      481 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      541 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      601 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      661 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      721 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      781 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      841 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctagccag
      901 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
      961 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     1021 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     1081 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
     1141 cctactctcg cgttaacgct agcatggatg ttttcccagt cacgacgttg taaaacgacg
     1201 gccagtctta agctcgggcc ccaaataatg attttatttt gactgatagt gacctgttcg
     1261 ttgcaacaaa ttgatgagca atgctttttt ataatgccaa ctttgtacaa aaaagcaggc
     1321 tcgctaaatt aaactaagcg ttattcgaga cgtggccgct ttaacgttac ctcaggtgcg
     1381 cgttcattat cagcgctccg taacaaacta ttcctgccct ggctgcctta caagatcacc
     1441 taattctctg ttgttttaca catccgtgtg cctgcattga cacttgcccc tcttctgtaa
     1501 agggggcttt tcttcctaaa gaatgccagg aaatcacact tccagtagca actaccgtca
     1561 cgtttaaaaa actctgactg cgggatgcta atttttttgt tcggctgttc taacttgtga
     1621 ttggctgctg gcagacgcgc cgcgctctga ttggctggag ccgctcctct ttgttgcggg
     1681 cgctctataa cagcatcctc ccttcattgc agagcagaaa ctcctcaagc acttcatttg
     1741 agcgatgtag agacacttct gctgcttctg acagatttag agtttggaga aaaaatcgcc
     1801 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1861 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1921 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1981 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2041 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2101 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2161 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2221 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2281 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2341 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2401 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2461 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2521 tccggactca gatctcgagc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg
     2581 ggatccaccg gatctagata actgataccc agctttcttg tacaaagttg gcattataag
     2641 aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat
     2701 tatttgccat ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     2761 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     2821 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     2881 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     2941 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     3001 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     3061 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     3121 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     3181 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     3241 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     3301 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     3361 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     3421 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     3481 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     3541 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     3601 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     3661 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     3721 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     3781 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatct
//
