LOCUS       sequence-178182-        4392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MultiSite Gateway entry clones, second fragment (attL5, attL2) 
            Firefly Luciferase CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4392)
  AUTHORS   Jackel C, Nogueira MS, Ehni N, Kraus C, Ranke J, Dohmann M, Noessner
            E, Nelson PJ
  TITLE     A vector platform for the rapid and efficient engineering of stable 
            complex transgenes.
  JOURNAL   Sci Rep. 2016 Oct 3;6:34365. doi: 10.1038/srep34365.
  PUBMED    27694838
REFERENCE   2  (bases 1 to 4392)
  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: "Sci Rep."; 
            date: "2016-10-3"; volume: "6:34365. doi"; pages: " 
            10.1038/srep34365"
COMMENT     Sequence Label: pENTR221-Firefly
FEATURES             Location/Qualifiers
     source          1..4392
                     /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    570..665
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      676..685
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             682..2334
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(748..766)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2375..2496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2386..2405)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2440..2459
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2504..2603)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2633..2652)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2634..2652)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2657..2673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2657..2673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2775..2794)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2786..3595
                     /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(2853..2872)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3742..4330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4231..4250
                     /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 tgtatacaaa
      661 agttgtattg gtaaagccac catggaagac gccaaaaaca taaagaaagg cccggcgcca
      721 ttctatccgc tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac
      781 gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac
      841 gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat
      901 acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg
      961 ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa
     1021 ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg
     1081 caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat
     1141 tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct
     1201 cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca
     1261 ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga
     1321 actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg
     1381 gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc
     1441 ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt
     1501 ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc
     1561 ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct
     1621 tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg
     1681 ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc
     1741 gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt
     1801 gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga
     1861 ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac
     1921 aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc
     1981 atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa
     2041 ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc
     2101 gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg
     2161 acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc
     2221 ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga
     2281 aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaattctag
     2341 agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     2401 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2461 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacccagc tttcttgtac
     2521 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
     2581 agtcaaaata aaatcattat ttgccatcca gctgcagggc ggccgcgata tcccctatag
     2641 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     2701 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     2761 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     2821 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     2881 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     2941 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     3001 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     3061 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     3121 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     3181 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     3241 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3301 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     3361 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3421 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     3481 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     3541 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     3601 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     3661 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     3721 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3781 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3841 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3901 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3961 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4021 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4081 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4141 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4201 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4261 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4321 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4381 tgctcacatg tt
//
