LOCUS       sequence-267253-        4222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone contains Firefly Luciferase2 
            without minimal promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4222)
  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 4222)
  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-luc2
FEATURES             Location/Qualifiers
     source          1..4222
                     /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      675..684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             681..2333
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     protein_bind    complement(2347..2446)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2463..2482)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2464..2482)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2487..2503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2487..2503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2605..2624)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2616..3425
                     /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(2683..2702)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3572..4160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4061..4080
                     /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 caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgg taaagccacc atggaagatg ccaaaaacat taagaagggc ccagcgccat
      721 tctacccact cgaagacggg accgccggcg agcagctgca caaagccatg aagcgctacg
      781 ccctggtgcc cggcaccatc gcctttaccg acgcacatat cgaggtggac attacctacg
      841 ccgagtactt cgagatgagc gttcggctgg cagaagctat gaagcgctat gggctgaata
      901 caaaccatcg gatcgtggtg tgcagcgaga atagcttgca gttcttcatg cccgtgttgg
      961 gtgccctgtt catcggtgtg gctgtggccc cagctaacga catctacaac gagcgcgagc
     1021 tgctgaacag catgggcatc agccagccca ccgtcgtatt cgtgagcaag aaagggctgc
     1081 aaaagatcct caacgtgcaa aagaagctac cgatcataca aaagatcatc atcatggata
     1141 gcaagaccga ctaccagggc ttccaaagca tgtacacctt cgtgacttcc catttgccac
     1201 ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga ccgggacaaa accatcgccc
     1261 tgatcatgaa cagtagtggc agtaccggat tgcccaaggg cgtagcccta ccgcaccgca
     1321 ccgcttgtgt ccgattcagt catgcccgcg accccatctt cggcaaccag atcatccccg
     1381 acaccgctat cctcagcgtg gtgccatttc accacggctt cggcatgttc accacgctgg
     1441 gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg cttcgaggag gagctattct
     1501 tgcgcagctt gcaagactat aagattcaat ctgccctgct ggtgcccaca ctatttagct
     1561 tcttcgctaa gagcactctc atcgacaagt acgacctaag caacttgcac gagatcgcca
     1621 gcggcggggc gccgctcagc aaggaggtag gtgaggccgt ggccaaacgc ttccacctac
     1681 caggcatccg ccagggctac ggcctgacag aaacaaccag cgccattctg atcacccccg
     1741 aaggggacga caagcctggc gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg
     1801 tggacttgga caccggtaag acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg
     1861 gccccatgat catgagcggc tacgttaaca accccgaggc tacaaacgct ctcatcgaca
     1921 aggacggctg gctgcacagc ggcgacatcg cctactggga cgaggacgag cacttcttca
     1981 tcgtggaccg gctgaagagc ctgatcaaat acaagggcta ccaggtagcc ccagccgaac
     2041 tggagagcat cctgctgcaa caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg
     2101 acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga
     2161 ccgagaagga gatcgtggac tatgtggcca gccaggttac aaccgccaag aagctgcgcg
     2221 gtggtgttgt gttcgtggac gaggtgccta aaggactgac cggcaagttg gacgcccgca
     2281 agatccgcga gattctcatt aaggccaaga agggcggcaa gatcgccgtg taataattct
     2341 agagtcaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     2401 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     2461 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     2521 ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga acaataaaac
     2581 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     2641 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2701 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     2761 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2821 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2881 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     2941 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     3001 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     3061 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     3121 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     3181 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     3241 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     3301 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     3361 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     3421 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     3481 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     3541 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3601 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3661 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3721 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3781 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3841 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3901 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3961 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4021 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4081 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4141 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4201 gctggccttt tgctcacatg tt
//
