LOCUS       sequence-291402-        8641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsGIGYF2-W288AF300AF306A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8641)
  AUTHORS   Peter D, Ruscica V, Bawankar P, Weber R, Helms S, Valkov E, Igreja 
            C, Izaurralde E
  TITLE     Molecular basis for GIGYF-Me31B complex assembly in 4EHP-mediated 
            translational repression.
  JOURNAL   Genes Dev. 2019 Oct 1;33(19-20):1355-1360. doi: 
            10.1101/gad.329219.119. Epub 2019 Aug 22.
  PUBMED    31439631
REFERENCE   2  (bases 1 to 8641)
  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: "Genes 
            Dev."; date: "2019-10-1"; volume: "33(19-20):1355-1360. doi"; pages:
            " 10.1101/gad.329219.119. Epub 2019 Aug 22"
COMMENT     Sequence Label: pT7-EGFP-C1-HsGIGYF2-W288AF300AF306A_AI
FEATURES             Location/Qualifiers
     source          1..8641
                     /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"
     primer_bind     613..632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      659..668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(710..731)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(971..990)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1318..1339
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3476..3487
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    5429..5550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5466..5485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5520..5539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5557..6012)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5694..5715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5906..5925
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6039..6143
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6145..6502
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6145..6336
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6168..6188)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6353..6488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6537..7331
                     /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             6537..7331
                     /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(6591..6610)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7201..7220
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7519..7538)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7563..7610
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7939..8527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8428..8447
                     /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 gcagagctct ctggctaact agagaaccca ctgcttactg
      601 gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcgc taccggtcgc
      661 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      721 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      781 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      841 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      901 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      961 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1021 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1081 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1141 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1201 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1261 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1321 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1381 gtccggactc agatctcgag ctatggcagc ggaaacgcag acactgaact ttgggcctga
     1441 atggctccga gctctgtcca gtggtgggag tattacatcc cctcctcttt ctccagcatt
     1501 gccgaagtat aaattagcag attatcgtta cggcagagaa gaaatgttag cacttttcct
     1561 taaagacaac aagatacctt cagaccttct ggataaagaa tttctgccta tcctccagga
     1621 ggaacccctt ccaccattgg ctctggtacc ctttacagaa gaagaacaga gaaacttttc
     1681 catgtctgta aatagtgctg ctgtcctgcg attgacagga cgaggaggag gaggaacagt
     1741 ggtgggggct cctagaggtc gaagttcttc aagagggcga ggcagaggca gaggtgaatg
     1801 tggtttctac caaagaagtt ttgatgaagt agagggtgtt tttggtcgag gaggtggcag
     1861 agaaatgcat agatcgcaga gctgggagga aaggggtgac agacgttttg aaaaaccagg
     1921 acgaaaagat gtagggagac caaattttga ggaaggtgga ccaacatcag tagggagaaa
     1981 gcatgaattt atacgctcag aaagtgaaaa ttggcgcatc tttagagagg aacaaaatgg
     2041 agaagatgaa gatggaggtt ggcgactagc tggatcaagg agggatggag agaggtggcg
     2101 acctcacagt cctgatggcc ctcgttctgc aggctggcgg gaacacatgg aacgacgtcg
     2161 gaggtttgag tttgattttc gagatagaga tgatgaacgg ggttaccgaa gggttcgctc
     2221 tggcagtggg agcatagatg atgacaggga tagcttgccc gaagcgtgct tagaggatgc
     2281 tgaagaagaa atgggtacag ccgactcatc tggagcagcc ctttctctaa aaaaagtaca
     2341 gaaagagcct attccagaag agcaggagat ggacttccgg cctgtggacg aaggggagga
     2401 gtgctctgac tctgagggta gccataatga agaggccaaa gaacccgata agacaaataa
     2461 gaaagaagga gagaaaacag atagagtagg agttgaagct agtgaggaaa ctccccagac
     2521 ctcatcatca tctgctagac caggtactcc ttcagaccat cagtctcagg aagcatcaca
     2581 gtttgagagg aaagatgaac caaaaactga gcaaacggaa aaagctgaag aggagactcg
     2641 gatggaaaat agtctaccag ccaaagtgcc cagcagaggg gatgaaatgg ttgctgatgt
     2701 ccagcagccc ctgtcgcaga ttccttcaga tacagcctct cctcttctca tacttccacc
     2761 tcctgttccc aatcctagtc ctactctccg gccagttgaa acaccagttg taggtgctcc
     2821 tggtatgggc agtgtttcca cagaacctga tgatgaagaa ggtctcaaac atttggagca
     2881 gcaagctgag aaaatggtgg cttatctcca agacagtgca ctagatgatg aaagattggc
     2941 atcaaaactg caagagcaca gagctaaagg agtgtcgatt ccattgatgc atgaagcaat
     3001 gcagaagtgg tattacaaag atcctcaggg agaaattcaa ggtcccttca ataatcagga
     3061 gatggcagaa tggtttcagg cgggctattt tactatgtct ttattggtga agagagcgtg
     3121 tgatgaaagc ttccaacctc ttggcgatat catgaaaatg tggggaaggg ttcccttttc
     3181 tccaggtcca gctccccctc ctcatatggg agagctggac caggaacgac tgaccaggca
     3241 gcaagaactc acagccttat accagatgca gcacctgcag taccagcagt ttttaataca
     3301 acaacaatat gcacaggttt tggcccaaca gcagaaagca gcactgtctt cccagcagca
     3361 gcagcagttg gcacttcttc ttcaacagtt tcagaccttg aagatgagaa tatctgatca
     3421 gaacatcatt ccctcagtaa ctaggtctgt gtccgtgcca gatactggct ctatctggga
     3481 gcttcagcca acagcttcac agcctacagt ttgggaaggt ggtagtgtat gggatcttcc
     3541 tctggacacc acgacaccag gccctgccct ggaacagctt cagcagctag agaaggccaa
     3601 agctgcaaag ctagagcaag agagaagaga ggcagaaatg agggcaaaac gggaagagga
     3661 agagcgaaag aggcaggaag aactccgaag acaacaggag gaaattcttc ggcgacagca
     3721 ggaagaagaa aggaaaaggc gagaggaaga agaacttgcc cgaaggaaac aggaagaggc
     3781 tctgcgtcgc cagcgggagc aagaaattgc attaaggcga cagcgagaag aggaagaaag
     3841 acagcagcaa gaagaagctc ttagaagact ggaagagagg agaagagaag aggaagaaag
     3901 gcggaagcag gaagaattgt tacgcaaaca ggaagaggag gctgcaaaat gggcccggga
     3961 agaagaagaa gcccagcgtc gattagagga gaaccggctg cggatggaag aggaggcagc
     4021 cagactccgg catgaggaag aagaacggaa gagaaaggag ctggaggtcc agcggcagaa
     4081 ggagttaatg cgccagaggc agcagcagca agaggctctc cggaggttgc agcagcagca
     4141 gcagcaacaa cagctggcgc agatgaagct tccttcttct tcaacgtggg gccagcagtc
     4201 caatacaaca gcatgtcagt cccaggccac gctgtcgttg gctgaaatcc aaaaactaga
     4261 ggaagaacga gaacggcagc ttcgagaaga gcaaaggcgc cagcagaggg agttgatgaa
     4321 agctcttcag cagcagcagc agcagcaaca gcagaaactc tcaggttggg ggaatgtcag
     4381 caaaccttca ggtaccacga aatctcttct ggagatccag caggaagagg ccaggcaaat
     4441 gcaaaagcag cagcagcagc agcagcaaca ccagcaacca aacagagctc gtaacaatac
     4501 gcattccaac ctgcacacca gcattgggaa ttctgtttgg ggctctataa atactggtcc
     4561 tcctaaccag tgggcatctg acctagtcag tagtatttgg agtaatgctg acactaaaaa
     4621 ctccaacatg ggattctggg atgatgcagt gaaagaggtg ggacctagga attcaacaaa
     4681 taaaaataaa aacaacgcca gtctcagtaa atctgtaggt gtgtctaacc ggcagaataa
     4741 gaaagtagaa gaagaagaaa agttgctgaa gctctttcag ggagtaaata aagcccaaga
     4801 tggatttacg cagtggtgtg aacagatgct tcatgccctt aatacggcaa ataacttgga
     4861 tgttcccaca tttgtttctt tcctgaaaga agtagaatct ccttatgagg tccatgatta
     4921 tatcagggcc tatttaggag atacttctga ggccaaggag tttgccaagc agttccttga
     4981 gcgccgtgcc aaacagaaag ccaaccagca gcgtcagcag cagcagctgc cgcagcagca
     5041 gcagcagccg ccacagcagc cgccacagca gccacaacag caggactctg tgtgggggat
     5101 gaaccacagt acactccatt cagtatttca gaccaatcaa agcaacaacc aacaatccaa
     5161 ttttgaggct gtgcagagtg gcaagaagaa gaaaaagcag aagatggtcc gagcagatcc
     5221 cagtttatta ggattttcag tcaatgcatc atcggagcga ctcaacatgg gtgaaatcga
     5281 gacgttggat gactactgag gatccaccgg atctagataa ctgatcataa tcagccatac
     5341 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     5401 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     5461 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5521 tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt
     5581 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5641 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5701 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     5761 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5821 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5881 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5941 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6001 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6061 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6121 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     6181 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     6241 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     6301 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     6361 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     6421 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     6481 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     6541 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     6601 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     6661 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     6721 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     6781 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     6841 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     6901 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     6961 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     7021 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     7081 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     7141 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     7201 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     7261 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     7321 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     7381 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     7441 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     7501 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     7561 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     7621 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     7681 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     7741 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     7801 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7861 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7921 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7981 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     8041 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     8101 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     8161 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     8221 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     8281 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     8341 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8401 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8461 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8521 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     8581 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     8641 t
//
