LOCUS       sequence-271894-        4280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone contains Firefly Luciferase2 
            insert driven by minimal promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4280)
  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 4280)
  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-minP:luc2
FEATURES             Location/Qualifiers
     source          1..4280
                     /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"
     promoter        675..706
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      733..742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             739..2391
                     /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(2405..2504)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2521..2540)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2522..2540)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2545..2561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2545..2561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2663..2682)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2674..3483
                     /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(2741..2760)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3630..4218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4119..4138
                     /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 agcaggctag acactagagg gtatataatg gaagctcgac ttccagcttg gcaatccggt
      721 actgttggta aagccaccat ggaagatgcc aaaaacatta agaagggccc agcgccattc
      781 tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa gcgctacgcc
      841 ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat tacctacgcc
      901 gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg gctgaataca
      961 aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc cgtgttgggt
     1021 gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga gcgcgagctg
     1081 ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa agggctgcaa
     1141 aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat catggatagc
     1201 aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca tttgccaccc
     1261 ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac catcgccctg
     1321 atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc gcaccgcacc
     1381 gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat catccccgac
     1441 accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac cacgctgggc
     1501 tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga gctattcttg
     1561 cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact atttagcttc
     1621 ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga gatcgccagc
     1681 ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca
     1741 ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat cacccccgaa
     1801 ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc taaggtggtg
     1861 gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc
     1921 cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct catcgacaag
     1981 gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca cttcttcatc
     2041 gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc agccgaactg
     2101 gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac
     2161 gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc
     2221 gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa gctgcgcggt
     2281 ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag
     2341 atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgta ataattctag
     2401 agtcacccag ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt
     2461 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc
     2521 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     2581 caaaatctct gatgttacat tgcacaagat aaaataatat catcatgaac aataaaactg
     2641 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     2701 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     2761 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     2821 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     2881 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     2941 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     3001 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     3061 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     3121 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     3181 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     3241 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     3301 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     3361 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     3421 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     3481 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac
     3541 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca
     3601 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3661 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3721 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3781 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3841 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3901 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3961 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4021 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4081 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4141 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4201 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4261 tggccttttg ctcacatgtt
//
