LOCUS       sequence-403974-        7541 bp    DNA     circular SYN 14-APR-2025
DEFINITION  expresses GPR35-FLAG-APEX in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7541)
  AUTHORS   Wyant GA, Yu W, Doulamis IP, Nomoto RS, Saeed MY, Duignan T, McCully
            JD, Kaelin WG Jr
  TITLE     Mitochondrial remodeling and ischemic protection by G 
            protein-coupled receptor 35 agonists.
  JOURNAL   Science. 2022 Aug 5;377(6606):621-629. doi: 10.1126/science.abm1638.
            Epub 2022 Aug 4.
  PUBMED    35926043
REFERENCE   2  (bases 1 to 7541)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.abm1638"; journalName: "Science"; date: "2022-08-5-
            5"; volume: "377"; issue: "6606"; pages: "621-629"
COMMENT     Sequence Label: pMXSIP-GPR35-FLAG-APEX
FEATURES             Location/Qualifiers
     source          1..7541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    678..1035
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1094..1510
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1440..1462
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1478..1494
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1520..1894
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1832..1848
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             2896..2919
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2920..3666
                     /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"
     misc_feature    3693..4259
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3709..4261
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3853..3870)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4080..4099
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4273..4875
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MVTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4772..4792
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5046..5515
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5645..5662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5816..6404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5896..5915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6568..7359,7360..7428))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7429..7533)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgca taatgaaaga ccccacctgt aggtttggca agctagctta
       61 agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaaa agttcagatc
      121 aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
      181 ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat
      241 atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg
      301 gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
      361 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
      421 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc
      481 tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc
      541 cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag
      601 cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca gggaccaccg
      661 acccaccacc gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg
      721 tctatgactg attttatgcg cctgcgtcgg tactagttag ctaactagct ctgtatctgg
      781 cggacccgtg gtggaactga cgagttcgga acacccggcc gcaaccctgg gagacgtccc
      841 agggacttcg ggggccgttt ttgtggcccg acctgagtcc aaaaatcccg atcgttttgg
      901 actctttggt gcacccccct aataggaggg atatgtggtt ctggtaggag acgagaacct
      961 aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tgggaccgaa gccgcgccgc
     1021 gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt
     1081 gtctgaaaat tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa
     1141 agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac
     1201 cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa
     1261 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
     1321 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
     1381 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
     1441 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
     1501 tctaggcgcc cccatatggc catatgagat cttatatggg gcacccccgc cccttgtaaa
     1561 cttccctgac cctgacatga caagagttac taacagcccc tctctccaag ctcacttaca
     1621 ggctctctac ttagtccagc acgaagtctg gagacctctg gcggcagcct accaagaaca
     1681 actggaccga ccggtggtac ctcaccctta ccgagtcggc gacacagtgt gggtccgccg
     1741 acaccagact aagaacctag aacctcgctg gaaaggacct tacacagtcc tgctgaccac
     1801 ccccaccgcc ctcaaagtag acggcatcgc agcttggata cacgccgccc acgtgaaggc
     1861 tgccgacccc gggggtggac catcctctag actgccggat ctagctagtt aattaaggat
     1921 cccagtgtgg tggtacggga attcgccacc atgaatggca cctacaacac ctgtggctcc
     1981 agcgacctca cctggccccc agcgatcaag ctgggcttct acgcctactt gggcgtcctg
     2041 ctggtgctag gcctgctgct caacagcctg gcgctctggg tgttctgctg ccgcatgcag
     2101 cagtggacgg agacccgcat ctacatgacc aacctggcgg tggccgacct ctgcctgctg
     2161 tgcaccttgc ccttcgtgct gcactccctg cgagacacct cagacacgcc gctgtgccag
     2221 ctctcccagg gcatctacct gaccaacagg tacatgagca tcagcctggt cacggccatc
     2281 gccgtggacc gctatgtggc cgtgcggcac ccgctgcgtg cccgcgggct gcggtccccc
     2341 aggcaggctg cggccgtgtg cgcggtcctc tgggtgctgg tcatcggctc cctggtggct
     2401 cgctggctcc tggggattca ggagggcggc ttctgcttca ggagcacccg gcacaatttc
     2461 aactccatgg cgttcccgct gctgggattc tacctgcccc tggccgtggt ggtcttctgc
     2521 tccctgaagg tggtgactgc cctggcccag aggccaccca ccgacgtggg gcaggcagag
     2581 gccacccgca aggctgcccg catggtctgg gccaacctcc tggtgttcgt ggtctgcttc
     2641 ctgcccctgc acgtggggct gacagtgcgc ctcgcagtgg gctggaacgc ctgtgccctc
     2701 ctggagacga tccgtcgcgc cctgtacata accagcaagc tctcagatgc caactgctgc
     2761 ctggacgcca tctgctacta ctacatggcc aaggagttcc aggaggcgtc tgcactggcc
     2821 gtggctcccc gtgctaaggc ccacaaaagc caggactctc tgtgcgtgac cctcgccgga
     2881 gggagcggag tcgacgacta caaggatgac gacgataagg gaaagtctta cccaactgtg
     2941 agtgctgatt accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct
     3001 gagaagagat gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac
     3061 aagggcacga agaccggtgg acccttcgga accatcaagc accctgccga actggctcac
     3121 agcgctaaca acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc
     3181 cctattttga gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg
     3241 ggtggaccta aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag
     3301 ggtcgcttgc ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct
     3361 atggggctta ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca
     3421 cacaaggagc gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac
     3481 tcatacttca cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac
     3541 aaggctcttt tgtctgaccc tgtattccgc cctctcgttg acaaatatgc agcggacgaa
     3601 gatgccttct ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct
     3661 gatgccgcgg cgtaagcggc cgctacgtaa attccgcccc cccccctaac gttactggcc
     3721 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3781 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3841 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     3901 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     3961 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     4021 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     4081 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     4141 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     4201 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     4261 atggccacaa ccatggttac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4321 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4381 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4441 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4501 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4561 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     4621 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     4681 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     4741 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     4801 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4861 aagcccggtg cctgagtcga cgataaaata aaagatttta tttagtctcc agaaaaaggg
     4921 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     4981 ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg
     5041 aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     5101 gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc
     5161 ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt
     5221 tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     5281 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     5341 tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc
     5401 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     5461 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcacatgc
     5521 agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa tggccatagt
     5581 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5641 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5701 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5761 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5821 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5881 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5941 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6001 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6061 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6121 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6181 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6241 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6301 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6361 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6421 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6481 gattatcaaa aaggatcttc acctagatcc ttttgcggcc ggccgcaaat caatctaaag
     6541 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6601 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6661 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6721 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6781 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6841 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6901 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6961 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7021 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7081 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7141 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7201 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7261 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7321 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7381 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7441 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7501 tatttagaaa aataaacaaa taggggttcc gcgcacattt c
//
