LOCUS       sequence-325536-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expresion of the B4E fusion protein, consists of the ampR gene 
            (encoding the enzyme beta-lactamase without the native signal 
            sequence) fused to residues 28-215 of the murine eIF4E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  AUTHORS   Moya-Ramirez I, Bouton C, Kontoravdi C, Polizzi K
  TITLE     High resolution biosensor to test the capping level and integrity of
            mRNAs.
  JOURNAL   Nucleic Acids Res. 2020 Dec 16;48(22):e129. doi: 
            10.1093/nar/gkaa955.
  PUBMED    33152073
REFERENCE   2  (bases 1 to 6730)
  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: "Nucleic 
            Acids Res."; date: "2020-12-16"; volume: "48(22):e129. doi"; pages: 
            " 10.1093/nar/gkaa955"
COMMENT     Sequence Label: pET28a-B4E
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(930..947)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     primer_bind     complement(1665..1683)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1679..1726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1763..2218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1850..1869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2060..2081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2310..3125)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2310..3125)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3033..3052
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3117..3136
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3247..3835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3736..3755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3989..4006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4021..4160
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4086..4105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4246..4268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4262..4453)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5262..6344)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6306..6325
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6345..6422)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6628..6647
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccaat gcaccaccac caccaccaca tgcatccgga
      121 aacactggtt aaagttaaag atgccgaaga tcagctgggt gcacgtgttg gttatattga
      181 actggatctg aatagcggca aaatcctgga aagttttcgt ccggaagaac gctttccgat
      241 gatgagcacc tttaaagttc tgctgtgtgg tgcagttctg agccgtgttg atgcaggtca
      301 agaacagctg ggacgtcgta ttcattatag ccagaatgat ctggttgaat atagtccggt
      361 taccgaaaaa catctgaccg atggtatgac cgttcgtgaa ctgtgtagcg cagcaattac
      421 catgagcgat aataccgcag caaatctgct gctgaccacc attggcggtc cgaaagaact
      481 gaccgcattt ctgcataata tgggtgatca tgttacccgt ctggatcgtt gggaacctga
      541 actgaatgaa gcaattccga atgatgaacg tgataccacc atgcctgcag caatggcaac
      601 cacactgcgt aaactgctga ccggtgaact gttaaccctg gcaagccgtc agcagctgat
      661 cgattggatg gaagcagata aagttgcagg tccgctgctg cgtagcgcac tgcctgcagg
      721 ttggtttatt gcagataaaa gcggtgccgg tgaacgtggt agccgtggta ttattgccgc
      781 actgggtcct gatggtaaac cgagccgtat tgttgtgatt tataccaccg gtagccaggc
      841 aaccatggat gaacgtaatc gtcagattgc agaaattggt gcaagcctga ttaaacattg
      901 gggtgctggt gccggtgcgg gtgccggtgc tggtgctggt gctggtgccg gtgcaggcgc
      961 aatggcaacc gttgaaccgg aaaccacacc gaccaccaat ccgcctccgg cagaagaaga
     1021 aaaaaccgaa agcaatcaag aagttgccaa tccggaacac tatatcaaac atccgctgca
     1081 gaatcgttgg gcactgtggt ttttcaaaaa cgacaaaagc aaaacctggc aggccaatct
     1141 gcgtctgatt agcaaatttg ataccgtgga agatttctgg gctctgtata atcatattca
     1201 gctgagcagc aatctgatgc ctggttgtga ttatagcctg tttaaagatg gtattgagcc
     1261 gatgtgggaa gatgaaaaaa acaaacgtgg tggtcgttgg ctgattaccc tgaatgcaca
     1321 gcagcgtcgt agcgatctgg atcgcttttg gctggaaacc ctgctgtgtc tgattggtga
     1381 aagtttcgat gattattcgg atgatgtttg tggtgccgtt gttaatgttc gtgcaaaagg
     1441 tgataaaatt gccatttgga ccaccgaatg tgaaaatcgt gatgcagtta cccatattgg
     1501 tcgcgtttat aaagaacgtc tgggtctgcc tccgaaaatt gttattggtt atcagagcca
     1561 tgcagatacc gcaaccaaaa gcggtagcac caccaaaaat cgttttgttg tttgagatcc
     1621 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1681 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1741 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1801 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1861 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1921 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1981 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2041 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2101 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2161 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2221 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2281 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2341 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2401 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2461 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2521 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2581 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2641 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     2701 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     2761 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     2821 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     2881 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     2941 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3001 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3061 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3121 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3181 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3241 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3301 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3361 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3421 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3481 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3541 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3601 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3661 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3721 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3781 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3841 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3901 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3961 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4021 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4081 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4141 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4201 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4261 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4321 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4381 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4441 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4501 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4561 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4621 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4681 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4741 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4801 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4861 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4921 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     4981 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5041 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5101 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5161 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5221 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5281 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5341 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5401 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5461 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5521 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5581 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5641 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5701 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     5761 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     5821 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     5881 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5941 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6001 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6061 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6121 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6181 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6241 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6301 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6361 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6421 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6481 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     6541 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     6601 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     6661 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     6721 cccgcgaaat
//
