LOCUS       sequence-370079-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the biotin proximity labeling enzyme MicroID2 with a 2X 
            mitochondrial targeting sequence (MTS) for expression and stable 
            integration into mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Johnson BS, Chafin L, Farkas D, Adair J, Elhance A, Farkas L, 
            Bednash JS, Londino JD
  TITLE     MicroID2: A Novel Biotin Ligase Enables Rapid Proximity Dependent 
            Proteomics.
  JOURNAL   Mol Cell Proteomics. 2022 Jun 7:100256. doi: 
            10.1016/j.mcpro.2022.100256.
  PUBMED    35688383
REFERENCE   2  (bases 1 to 6912)
  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: "Mol Cell 
            Proteomics. 2022 Jun 7:100256. doi: 10.1016/j.mcpro.2022.100256."
COMMENT     Sequence Label: pSBbi_RP_2XMTS-MicroID2-FLAG
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             535..1236
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MLSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(668..686)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     936..955
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1130..1153
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1246..1302
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1303..1902
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDXVPRXVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1303..1322)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1799..1819
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2002..2021)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2067..2122
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2121..2140)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2514..2531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2685..3273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2765..2784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3444..4235,4236..4304))
                     /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"
     primer_bind     4067..4086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4305..4409)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4841..5065
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5054..5071
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5103..5126)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(5679..5753)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(5775..5849)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(5926..5946)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 gccctgcagc cttcagtcgc ttgaaggggt aatggcgctt ccactcacaa acatggcgga
       61 cagagcgtgt gaacgagatg aacagcccct caaaaatatg gccgccgagg ctggacggcc
      121 gtgccccagc agcaccgcct ccgcgcccca cgtgatctct cgccgggcac agcgctgacc
      181 gcggaggtcc aaccggaaga atgtccggat tggacattcg gaagagggcc cgccttccct
      241 ggggaatctc tgcgcacgcg cagaacrctt cgaccaatga aaacacagga agccgtccgc
      301 gcaaccgcgt tgcgtcactt ctgccgcccc tgtttcaagg tatatagccg tagacggaac
      361 ttcgcctttc tctcggcctt agcgccattt ttttgggtga gtgttttttg gttcctgcgt
      421 tgggattccg tgtacaatcc atagacatct gacctcggca cttagcatca tcacagcaaa
      481 ctaactgtag cctttctctc tttccctgta gaaacctctg cacctgaggc caccatgttg
      541 agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc
      601 atgaacggcc atgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      661 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
      721 tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat
      781 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      841 ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag
      901 gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagacaatg
      961 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
     1021 caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac
     1081 atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc
     1141 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac
     1201 cacctgttcc tgtacggcat ggacgagctg tacaagggca gtggagctac taacttcagc
     1261 ctgctgaagc aggctggtga cgtcgaggag aatcctggcc ccatgaccga gtacaagccc
     1321 acggtgcgcc tcgccacccg cgacgmcgtc cccaggnccg tacgcaccct cgccgccgcg
     1381 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     1441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     1501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     1561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     1621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     1681 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     1741 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     1801 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     1861 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gattcgaagg cctgtcgtga
     1921 attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     1981 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     2041 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     2101 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     2161 tcagaatgag tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac
     2221 tagctactcg ggacccctta ccgaaacatc gccgcattct gcagaggagt cgagtgtatg
     2281 taaacttctg acccactggg aatgtgatga aagaaataaa agctgaaatg aatcattctc
     2341 tctactatta ttctgatatt tcacattctt aaaataaagt ggtgatccta actgacctaa
     2401 gacagggaat ttttactagg attaaatgtc aggaattgtg aaaaagtgag tttaaatgta
     2461 tttggctaag gtgtatgtaa acttccgact tcaactgtat agggatccgc ttcctcgctc
     2521 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2641 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2701 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2761 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2821 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2881 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2941 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3001 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3061 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3121 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3181 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3241 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3301 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3361 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3421 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3481 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3541 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3601 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3661 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3721 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3781 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3841 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3901 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3961 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4021 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4081 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4141 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4201 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4261 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4321 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4381 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     4441 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     4501 aatattttgt taaaattgga tccctataca gttgaagtcg gaagtttaca tacacttaag
     4561 ttggagtcat taaaactcgt ttttcaacta ctccacaaat ttcttgttaa caaacaatag
     4621 ttttggcaag tcagttagga catctacttt gtgcatgaca caagtcattt ttccaacaat
     4681 tgtttacaga cagattattt cacttataat tcactgtatc acaattccag tgggtcagaa
     4741 gtttacatac actaagttcg actcctctgc agaatgcggc gatgtttcgg taaggggtcc
     4801 gctatctagt aggcccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc
     4861 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
     4921 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
     4981 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
     5041 gatggctggc aactagaagg cacagtcgag gctgatcagc gagctctaga ctattaggta
     5101 cccttgtcgt catcgtcttt gtagtcgcta gcctccttga acttcttcag gttctcgctg
     5161 attctcttca gcaccttcag cagcacctcc tttctgtccc agtccttgcc ggtgatctcg
     5221 tacagggtgg tggctctgtc cttgatctcc tcggggatct ctctctggtt cacgttgatg
     5281 ccgatgccca cgatcagctt gtccttgctc agctcgcaca gcacgccgct cacctttctc
     5341 tcctggaagt acacgtcgtt gggccacttc aggctgaagg ggatctcggt gatctcctcc
     5401 agggcctcgc tcacgctcag gcccagcacc aggggcagct gcagcaggtt ctcgaactcc
     5461 ttggggttca gcaggaagct gaagtacagg ccgccctcct ggctaaacca ccttctgccc
     5521 gagccgcctc tgcccctggt ctgtctgtcg gccaccaggg cggtgccgta gctcacgttc
     5581 cactccttca gtctctcctg ggtsctgtcc acctccttca gccagatcag gttcttgaag
     5641 tcgacctgca gaattcgaag cttccccaac gaatggatct tggcgcgcgg cactgggagc
     5701 cgccgggccg agcctgtcaa gccccgcagc agcagcggcg tcaggacgga catgggatcc
     5761 cccaacgaat ggatcttggc gcgcggcact gggagccgcc gggccgagcc tgtcaagccc
     5821 cgcagcagca gcggcgtcag gacggacata agcttgggtc tccctggcct cagaggccag
     5881 cttggggtag ttttcacgac accttaaatg gaagaaaaaa actttgaacc actgtctgag
     5941 gcttgagaat gaaccaagat ccaaactcaa aaagggcaaa ttccaaggag aattacatca
     6001 agtgccaagc tggcctaact tcagtctcca cccactcagt atggggaaac tccatcgcat
     6061 aaaacccctc cccccaacct aaagacgacg tactccaaaa gctcgagaac taatcgaggt
     6121 gcctggacgg cgcccggtac tccgtggagt cacatgaagc gacggctgag gacggaaagg
     6181 cccttttcct ttgtgtgggt gactcacccg cccgctctcc cgagcgccgc gtcctccatt
     6241 ttgagctccc tgcagcaggg ccgggaagcg gccatctttc cgctcacgca actggtgccg
     6301 accgggccag ccttgccgcc cagggcgggg cgatacacgg cggcgcgagg ccaggcacca
     6361 gagcaggccg gccagcttga gactaccccc gtccgattct cggtggccgc gctcgcaggc
     6421 cccgcctcgc cgaacatgtg cgctgggacg cacgggcccc gtcgccgccc gcggccccaa
     6481 aaaccgaaat accagtgtgc agatcytggc ccgcatttac aagactatct tgccagaaaa
     6541 aaagcgtcgc agcaggtcat caaaaatttt aaatggctag agacttatcg aaagcagcga
     6601 gacaggcgcg aaggtgccac cagattcgca cgcggcggcc ccagcgccca agccaggcct
     6661 caactcaagc acgaggcgaa ggggctcctt aagcgcaagg cctcgaactc tcccacccac
     6721 ttccaacccg aagctcggga tcaagaatca cgtactgsag ccaggggcgt ggaagtaatt
     6781 caaggcacgc aagggccata acccgtaaag aggccaggcc cgcgggaacc acacacggca
     6841 cttacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     6901 cttatatacg gc
//
