LOCUS       sequence-486896-        6572 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Mammalian Expression of 7BoxB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6572)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 6572)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pEGFP-N3-FLuc-7BoxB_O
FEATURES             Location/Qualifiers
     source          1..6572
                     /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)"
     protein_bind    3255..3273
                     /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    3279..3297
                     /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    3432..3553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3469..3488)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3523..3542
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3560..4015)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3697..3718)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3909..3928
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4042..4146
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4171..4191)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4215..4425
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4237..4433
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4284..4419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4468..5262
                     /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             4468..5262
                     /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(4522..4541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5132..5151
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5450..5469)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5494..5541
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5870..6458
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6359..6378
                     /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 tcgactaagt
     3241 ccaactacta aactgggccc tgaagaaggg cccatatagg gccctgaaga agggccctat
     3301 cgaggatatt atctcgagga ctctagatca taatcagcca taccacattt gtagaggttt
     3361 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3421 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3481 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3541 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3601 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3661 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3721 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3781 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3841 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3901 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3961 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     4021 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4081 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4141 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     4201 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4261 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4321 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4381 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     4441 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4501 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4561 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4621 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     4681 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4741 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4801 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4861 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4921 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4981 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5041 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5101 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5161 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5221 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5281 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5341 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5401 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     5461 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     5521 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     5581 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     5641 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     5701 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5761 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5821 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5881 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5941 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6001 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6061 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6121 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6181 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6241 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6301 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6361 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6421 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6481 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6541 attctgtgga taaccgtatt accgccatgc at
//
