LOCUS       sequence-338035-        6654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2_MSP1D1 in Bl21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6654)
  AUTHORS   Bao H
  TITLE     Developing Nanodisc-ID for label-free characterizations of membrane 
            proteins.
  JOURNAL   Commun Biol. 2021 Apr 30;4(1):514. doi: 10.1038/s42003-021-02043-y.
  PUBMED    33931748
REFERENCE   2  (bases 1 to 6654)
  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: "Commun 
            Biol."; date: "2021-04-30"; volume: "4(1):514. doi"; pages: " 
            10.1038/s42003-021-02043-y"
COMMENT     Sequence Label: 28A_APEX2_MSP1D1
FEATURES             Location/Qualifiers
     source          1..6654
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             757..1503
                     /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="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1519..1536
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1589..1607)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1603..1650
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1687..2142
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1774..1793)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1984..2005
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2234..3049)
                     /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(2234..3049)
                     /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     2957..2976
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3041..3060
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3171..3759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3660..3679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3913..3930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3945..4084
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4010..4029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4170..4192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4186..4377)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5186..6268)
                     /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     6230..6249
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6269..6346)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6552..6571
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatcct ctaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
      241 tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa agatttagaa
      301 gaggtgaagg ccaaggttca gccatatctc gatgactttc agaaaaaatg gcaggaagag
      361 atggaattat atcgtcaaaa ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc
      421 caaaaactcc atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
      481 gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga tgaacttcgc
      541 cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg cttggctgag
      601 taccacgcga aagcgacaga acacctgagc accttgagcg aaaaagcgaa accggcgctg
      661 gaagatctac gccagggctt attgcctgtt cttgagagct ttaaagtcag ttttctgtca
      721 gctctggaag aatatactaa aaagctgaat acccagggaa agtcttaccc aactgtgagt
      781 gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt catcgctgag
      841 aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac ctttgacaag
      901 ggcacgaaga ccggtggacc cttcggaacc atcaagcacc ctgccgaact ggctcacagc
      961 gctaacaacg gtcttgacat cgctgttagg cttttggagc cactcaaggc ggagttccct
     1021 attttgagct acgccgattt ctaccagttg gctggcgttg ttgccgttga ggtcacgggt
     1081 ggacctaagg ttccattcca ccctggaaga gaggacaagc ctgagccacc accagagggt
     1141 cgcttgcccg atcccactaa gggttctgac catttgagag atgtgtttgg caaagctatg
     1201 gggcttactg accaagatat cgttgctcta tctgggggtc acactattgg agctgcacac
     1261 aaggagcgtt ctggatttga gggtccctgg acctctaatc ctcttatttt cgacaactca
     1321 tacttcacgg agttgttgag tggtgagaag gaaggtctcc ttcagctacc ttctgacaag
     1381 gctcttttgt ctgaccctgt attccgccct ctcgttgaca aatatgcagc ggacgaagat
     1441 gccttctttg ctgattacgc tgaggctcac caaaagcttt ccgagcttgg gtttgctgat
     1501 gcctaagccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1561 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1621 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1681 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1741 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1801 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1861 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1921 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1981 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2041 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2101 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2161 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2221 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     2281 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     2341 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     2401 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     2461 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     2521 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     2581 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     2641 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     2701 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     2761 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2821 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2881 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2941 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3001 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     3061 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     3121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3781 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3901 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3961 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4021 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4081 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4141 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4201 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4261 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4321 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4381 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4441 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4501 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4561 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4621 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4681 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4741 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4801 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4861 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4921 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4981 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5041 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5101 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5161 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5221 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5281 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5341 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5401 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5461 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5521 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5581 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5641 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5701 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5761 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5821 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5881 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5941 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6001 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6061 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6121 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6181 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6241 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6301 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6361 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6421 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6481 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6541 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6601 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
