LOCUS       sequence-254973-        7477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2 fusion to DEAD-box translation factor eIF4A1 in 
            bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  AUTHORS   Padron A, Iwasaki S, Ingolia NT
  TITLE     Proximity RNA Labeling by APEX-Seq Reveals the Organization of 
            Translation Initiation Complexes and Repressive RNA Granules.
  JOURNAL   Mol Cell. 2019 Aug 22;75(4):875-887.e5. doi: 
            10.1016/j.molcel.2019.07.030.
  PUBMED    31442426
REFERENCE   2  (bases 1 to 7477)
  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"; doi: 
            "10.1016/j.molcel.2019.07"; journalName: "Mol Cell"; date: 
            "2019-08-22- 22"; volume: "75"; issue: "4"; pages: "875-887.e5"
COMMENT     Sequence Label: pHis APEX2-eIF4A1
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3491..3510
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3491..3509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3510..3534
                     /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             3549..3571
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3590..3607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3608..4357
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     CDS             4364..4390
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             4394..4414
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(5655..5679)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5693..5710
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5763..5781)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5777..5824
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5861..6316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5948..5967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6158..6179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6342..6446
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6447..6515,6516..7307)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6665..6684)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcc agatctcgat
     3481 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     3541 gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca
     3601 tcatcacatg ggaaagtctt acccaactgt gagtgctgat taccaggacg ccgttgagaa
     3661 ggcgaagaag aagctcagag gcttcatcgc tgagaagaga tgcgctcctc taatgctccg
     3721 tttggcattc cactctgctg gaacctttga caagggcacg aagaccggtg gacccttcgg
     3781 aaccatcaag caccctgccg aactggctca cagcgctaac aacggtcttg acatcgctgt
     3841 taggcttttg gagccactca aggcggagtt ccctattttg agctacgccg atttctacca
     3901 gttggctggc gttgttgccg ttgaggtcac gggtggacct aaggttccat tccaccctgg
     3961 aagagaggac aagcctgagc caccaccaga gggtcgcttg cccgatccca ctaagggttc
     4021 tgaccatttg agagatgtgt ttggcaaagc tatggggctt actgaccaag atatcgttgc
     4081 tctatctggg ggtcacacta ttggagctgc acacaaggag cgttctggat ttgagggtcc
     4141 ctggacctct aatcctctta ttttcgacaa ctcatacttc acggagttgt tgagtggtga
     4201 gaaggaaggt ctccttcagc taccttctga caaggctctt ttgtctgacc ctgtattccg
     4261 ccctctcgtt gacaaatatg cagcggacga agatgccttc tttgctgatt acgctgaggc
     4321 tcaccaaaag ctttccgagc ttgggtttgc tgatgccctg cagctgcctc ccctggagcg
     4381 cctgaccctg gacgaaaacc tgtattttca gggatccatg tctgcgagcc aggattcccg
     4441 atccagagac aatggccccg atgggatgga gcccgaaggc gtcatcgaga gtaactggaa
     4501 tgagattgtt gacagctttg atgacatgaa cctctcggag tcccttctcc gtggcatcta
     4561 cgcctatggt tttgagaagc cctctgccat ccagcagcga gccattctac cttgtatcaa
     4621 gggttatgat gtgattgctc aagcccaatc tgggactggg aaaacggcca catttgccat
     4681 atcgattctg cagcagattg aattagatct aaaagccacc caggccttgg tcctagcacc
     4741 cactcgagaa ttggctcagc agatacagaa ggtggtcatg gcactaggag actacatggg
     4801 cgcctcctgt cacgcctgta tcgggggcac caacgtgcgt gctgaggtgc agaaactgca
     4861 gatggaagct ccccacatca tcgtgggtac ccctggccgt gtgtttgata tgcttaaccg
     4921 gagatacctg tcccccaaat acatcaagat gtttgtactg gatgaagctg acgaaatgtt
     4981 aagccgtgga ttcaaggacc agatctatga catattccaa aagctcaaca gcaacaccca
     5041 ggtagttttg ctgtcagcca caatgccttc tgatgtgctt gaggtgacca agaagttcat
     5101 gagggacccc attcggattc ttgtcaagaa ggaagagttg accctggagg gtatccgcca
     5161 gttctacatc aacgtggaac gagaggagtg gaagctggac acactatgtg acttgtatga
     5221 aaccctgacc atcacccagg cagtcatctt catcaacacc cggaggaagg tggactggct
     5281 caccgagaag atgcatgctc gagatttcac tgtatccgcc atgcatggag atatggacca
     5341 aaaggaacga gacgtgatta tgagggagtt tcgttctggc tctagcagag ttttgattac
     5401 cactgacctg ctggccagag gcattgatgt gcagcaggtt tctttagtca tcaactatga
     5461 ccttcccacc aacagggaaa actatatcca cagaatcggt cgaggtggac ggtttggccg
     5521 taaaggtgtg gctattaaca tggtgacaga agaagacaag aggactcttc gagacattga
     5581 gaccttctac aacacctcca ttgaggaaat gcccctcaat gttgctgacc tcatctagaa
     5641 gggtgggcgc gccgacccag ctttcttgta caaagtggtg atgtacctcg agcaccacca
     5701 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     5761 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     5821 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     5881 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5941 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6001 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6061 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6121 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6181 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     6241 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6301 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6361 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6421 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6481 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6541 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6601 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6661 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6721 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6781 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6841 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6901 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6961 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     7021 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     7081 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     7141 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     7201 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     7261 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     7321 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     7381 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7441 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
