LOCUS       sequence-314280-        6734 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 6734)
  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 6734)
  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..6734
                     /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(2311..3126)
                     /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(2311..3126)
                     /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     3034..3053
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3118..3137
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3248..3836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3737..3756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3990..4007
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4022..4164
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4090..4109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4250..4272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4266..4457)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5266..6348)
                     /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     6310..6329
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6349..6426)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6632..6651
                     /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 tattaacgtt tacaatttca
     2221 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2281 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     2341 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2401 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2461 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2521 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2581 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2641 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2701 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2761 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2821 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2881 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2941 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     3001 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     3061 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3121 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     3181 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3241 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3301 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3361 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3421 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3481 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3541 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3601 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3661 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3721 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3781 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3841 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3901 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3961 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4021 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     4081 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4141 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4201 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4261 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4321 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4381 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4441 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4501 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4561 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4621 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4681 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4741 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4801 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4861 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4921 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4981 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5041 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5101 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5161 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5221 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5281 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5341 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5401 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5461 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5521 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5581 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5641 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5701 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5761 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5821 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5881 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5941 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6001 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6061 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6121 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6181 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6241 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6301 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6361 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6421 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6481 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6541 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6601 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6661 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6721 cgatcccgcg aaat
//
