LOCUS       sequence-355330-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of 5BoxB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 6561)
  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"
COMMENT     Sequence Label: pEGFP-N3-FLuc-5BoxB_O
FEATURES             Location/Qualifiers
     source          1..6561
                     /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"
     regulatory      2265..2274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2271..2273,2274..2276,2277..2990)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2271..2273,2274..2276,2277..2987)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2316..2337)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2577..2596)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2924..2945
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3045..3063
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3089..3107
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3113..3131
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3172..3190
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3196..3214
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     polyA_signal    3349..3470
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3386..3405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3440..3459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3477..3932)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3614..3635)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3826..3845
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3959..4063
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4088..4108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4204..4414
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4226..4422
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4273..4408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4457..5251
                     /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             4457..5251
                     /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(4511..4530)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5121..5140
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5439..5458)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5483..5530
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5859..6447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6348..6367
                     /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 catcgccacc atggtgagca
     2281 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2341 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2401 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2461 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2521 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2581 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2641 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2701 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2761 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2821 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2881 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2941 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg
     3001 agcagtaatt ctagataagt ccaactacta aactggggat tcctgggccc tgaagaaggg
     3061 cccctcgact aagtccaact actaaactgg gccctgaaga agggcccata tagggccctg
     3121 aagaagggcc ctatcgagga tattatctcg actaagtcca actactaaac tgggccctga
     3181 agaagggccc atatagggcc ctgaagaagg gccctatcga ggatattatc tcgaggactc
     3241 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3301 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3361 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3421 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     3481 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3541 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3601 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3661 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3721 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3781 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3841 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3901 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     3961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4021 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     4081 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4141 agtatgcaaa gcatgcatct caattagtcw smrwymrkrk kyssrmmsyy cccaggctcc
     4201 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4261 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4321 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4381 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4441 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4501 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4561 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4621 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4681 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4741 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4801 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4861 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4921 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4981 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5041 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5101 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5161 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5221 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5281 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5341 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5401 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5461 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5521 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5581 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5641 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5701 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6001 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6121 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6481 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6541 aaccgtatta ccgccatgca t
//
