LOCUS       sequence-489476-        6747 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Mammalian Expression of K21-Rluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6747)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 6747)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pEGFP-N3-FLuc-K21-Rluc_AH
FEATURES             Location/Qualifiers
     source          1..6747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             606..2255
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEYAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(672..690)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             2262..2303
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2313..2369
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2445..2471
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2478..3413
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    3535..3656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3572..3591)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3626..3645
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3663..4118)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3800..3821)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4012..4031
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4145..4249
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4251..4608
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4251..4442
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4274..4294)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4459..4594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4643..5437
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4643..5437
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4697..4716)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5307..5326
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5625..5644)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5669..5716
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6045..6633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6534..6553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacag
      601 ccaccatgga atacgccaaa aacataaaga aaggcccggc gccattctat ccgctggaag
      661 atggaaccgc tggagagcaa ctgcataagg ctatgaagag atacgccctg gttcctggaa
      721 caattgcttt tacagatgca catatcgagg tggacatcac ttacgctgag tacttcgaaa
      781 tgtccgttcg gttggcagaa gctatgaaac gatatgggct gaatacaaat cacagaatcg
      841 tcgtatgcag tgaaaactct cttcaattct ttatgccggt gttgggcgcg ttatttatcg
      901 gagttgcagt tgcgcccgcg aacgacattt ataatgaacg tgaattgctc aacagtatgg
      961 gcatttcgca gcctaccgtg gtgttcgttt ccaaaaaggg gttgcaaaaa attttgaacg
     1021 tgcaaaaaaa gctcccaatc atccaaaaaa ttattatcat ggattctaaa acggattacc
     1081 agggatttca gtcgatgtac acgttcgtca catctcatct acctcccggt tttaatgaat
     1141 acgattttgt gccagagtcc ttcgataggg acaagacaat tgcactgatc atgaactcct
     1201 ctggatctac tggtctgcct aaaggtgtcg ctctgcctca tagaactgcc tgcgtgagat
     1261 tctcgcatgc cagagatcct atttttggca atcaaatcat tccggatact gcgattttaa
     1321 gtgttgttcc attccatcac ggttttggaa tgtttactac actcggatat ttgatatgtg
     1381 gatttcgagt cgtcttaatg tatagatttg aagaagagct gtttctgagg agccttcagg
     1441 attacaagat tcaaagtgcg ctgctggtgc caaccctatt ctccttcttc gccaaaagca
     1501 ctctgattga caaatacgat ttatctaatt tacacgaaat tgcttctggt ggcgctcccc
     1561 tctctaagga agtcggggaa gcggttgcca agaggttcca tctgccaggt atcaggcaag
     1621 gatatgggct cactgagact acatcagcta ttctgattac acccgagggg gatgataaac
     1681 cgggcgcggt cggtaaagtt gttccatttt ttgaagcgaa ggttgtggat ctggataccg
     1741 ggaaaacgct gggcgttaat caaagaggcg aactgtgtgt gagaggtcct atgattatgt
     1801 ccggttatgt aaacaatccg gaagcgacca acgccttgat tgacaaggat ggatggctac
     1861 attctggaga catagcttac tgggacgaag acgaacactt cttcatcgtt gaccgcctga
     1921 agtctctgat taagtacaaa ggctatcagg tggctcccgc tgaattggaa tccatcttgc
     1981 tccaacaccc caacatcttc gacgcaggtg tcgcaggtct tcccgacgat gacgccggtg
     2041 aacttcccgc cgccgttgtt gttttggagc acggaaagac gatgacggaa aaagagatcg
     2101 tggattacgt cgccagtcaa gtaacaaccg cgaaaaagtt gcgcggagga gttgtgtttg
     2161 tggacgaagt accgaaaggt cttaccggaa aactcgacgc aagaaaaatc agagagatcc
     2221 tcataaaggc caagaagggc ggaaagatcg ccgtgggatc cggtaagcct atccctaacc
     2281 ctctcctcgg tctcgattct acgggaagcg gagctactaa cttcagcctg ctgaagcagg
     2341 ctggagacgt ggaggagaac cctggaccta agcttaaaaa aaaaaaaaaa aaaaaaaaaa
     2401 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaact cgagtaccca tacgatgttc
     2461 cagattacgc tgaattcatg gcttccaagg tgtacgaccc cgagcaacgc aaacgcatga
     2521 tcactgggcc tcagtggtgg gctcgctgca agcaaatgaa cgtgctggac tccttcatca
     2581 actactatga ttccgagaag cacgccgaga acgccgtgat ttttctgcat ggtaacgctg
     2641 cctccagcta cctgtggagg cacgtcgtgc ctcacatcga gcccgtggct agatgcatca
     2701 tccctgatct gatcggaatg ggtaagtccg gcaagagcgg gaatggctca tatcgcctcc
     2761 tggatcacta caagtacctc accgcttggt tcgagctgct gaaccttcca aagaaaatca
     2821 tctttgtggg ccacgactgg ggggcttgtc tggcctttca ctactcctac gagcaccaag
     2881 acaagatcaa ggccatcgtc catgctgaga gtgtcgtgga cgtgatcgag tcctgggacg
     2941 agtggcctga catcgaggag gatatcgccc tgatcaagag cgaagagggc gagaaaatgg
     3001 tgcttgagaa taacttcttc gtcgagacca tgctcccaag caagatcatg cggaaactgg
     3061 agcctgagga gttcgctgcc tacctggagc cattcaagga gaagggcgag gttagacggc
     3121 ctaccctctc ctggcctcgc gagatccctc tcgttaaggg aggcaagccc gacgtcgtcc
     3181 agattgtccg caactacaac gcctaccttc gggccagcga cgatctgcct aagatgttca
     3241 tcgagtccga ccctgggttc ttttccaacg ctattgtcga gggagctaag aagttcccta
     3301 acaccgagtt cgtgaaggtg aagggcctcc acttcagcca ggaggacgct ccagatgaaa
     3361 tgggtaagta catcaagagc ttcgtggagc gcgtgctgaa gaacgagcag taagcggccg
     3421 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3481 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3541 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3601 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3661 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3721 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3781 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3841 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3901 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3961 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4021 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4081 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4141 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4201 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4261 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4321 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4381 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4441 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4501 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4561 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4621 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4681 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4741 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4801 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4861 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4921 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4981 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5041 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5101 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5161 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5221 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5281 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5341 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5401 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5461 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5521 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5581 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5641 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5701 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5761 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5821 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5881 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5941 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6001 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6061 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6181 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6661 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6721 gtggataacc gtattaccgc catgcat
//
