LOCUS       sequence-372835-        7173 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 7173)
  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 7173)
  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..7173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        109..213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(214..282,283..1074)
                     /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     complement(432..451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1245..1833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1734..1753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1987..2004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2378..2397
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2396..2451
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     2497..2516
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2616..3215)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2699..2719)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3196..3215
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(3216..3272)
                     /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             complement(3282..3983)
                     /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(3365..3388)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3563..3582)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3832..3850
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        4615..5797
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     5745..5765
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5842..5916
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             5938..6012
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             6565..6588
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6620..6637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6626..6850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
       61 tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct
      121 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      181 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      241 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
      301 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
      361 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
      421 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
      481 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
      541 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
      601 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
      661 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      721 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      781 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      841 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      901 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      961 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1021 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1081 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1141 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1201 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1261 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1321 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1381 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1441 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1501 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1561 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1621 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1681 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1741 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1801 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1861 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1921 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     1981 gagcgcagcg agtcagtgag cgaggaagcg gatccctata cagttgaagt cggaagttta
     2041 catacacctt agccaaatac atttaaactc actttttcac aattcctgac atttaatcct
     2101 agtaaaaatt ccctgtctta ggtcagttag gatcaccact ttattttaag aatgtgaaat
     2161 atcagaataa tagtagagag aatgattcat ttcagctttt atttctttca tcacattccc
     2221 agtgggtcag aagtttacat acactcgact cctctgcaga atgcggcgat gtttcggtaa
     2281 ggggtcccga gtagctagtt catggcagcc agcatatggc atatgttgcc aaactctaaa
     2341 ccaaatactc attctgatgt tttaaatgat ttgccctccc atatgtcctt ccgagtgaga
     2401 gacacaaaaa attccaacac actattgcaa tgaaaataaa tttcctttat tagccagaag
     2461 tcagatgctc aaggggcttc atgatgtccc cataattttt ggcagaggga aaaagatctc
     2521 agtggtattt gtgagccagg gcattggcca caccagccac caccttctga taggcagcct
     2581 gcacctgagg agtgaattca cgacaggcct tcgaatcagg caccgggctt gcgggtcatg
     2641 caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc
     2701 tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac cccggcgcgc
     2761 tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc ctggtggtcg
     2821 ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg cggcgccagg
     2881 aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc catgcgcggg
     2941 ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca gaccgccacc
     3001 gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag
     3061 agttcttgca gctcggtgac ccgctcgatg tggcggtccg gatcgacggt gtggcgcgtg
     3121 gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg ccctggggac gtcgtcgcgg
     3181 gtggcgaggc gcaccgtggg cttgtactcg gtcatggggc caggattctc ctcgacgtca
     3241 ccagcctgct tcagcaggct gaagttagta gctccactgc ccttgtacag ctcgtccatg
     3301 ccgtacagga acaggtggtg gcggccctcg gagcgctcgt actgttccac gatggtgtag
     3361 tcctcgttgt gggaggtgat gtccagcttg gtgtccacgt agtagtagcc gggcagttgc
     3421 acgggcttct tggccatgta gatggtcttg aactccacca ggtagtggcc gccgtccttc
     3481 agcttcaggg cctggtggat ctcgcccttc agcacgccgt cgcgggggta caggcgctcg
     3541 gtggaggcct cccagcccat tgtcttcttc tgcattacgg ggccgtcggg ggggaagttg
     3601 gtgccgcgca tcttcacctt gtagatcagc gtgccgtcct gcagggagga gtcctgggtc
     3661 acggtcacca gaccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg
     3721 aaggacagct tcttgtaatc ggggatgtcg gcggggtgct tcacgtacgc cttggagccg
     3781 tacatgaact ggggggacag gatgtcccag gcgaagggca gggggccgcc cttggtcacc
     3841 ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg
     3901 aactcatggc cgttcatgga gccctccatg cgcaccttga agcgcatgaa ctctttgatg
     3961 acctcctcgc ccttgctcaa catggtggcc tcaggtgcag aggtttctac agggaaagag
     4021 agaaaggcta cagttagttt gctgtgatga tgctaagtgc cgaggtcaga tgtctatgga
     4081 ttgtacacgg aatcccaacg caggaaccaa aaaacactca cccaaaaaaa tggcgctaag
     4141 gccgagagaa aggcgaagtt ccgtctacgg ctatatacct tgaaacaggg gcggcagaag
     4201 tgacgcaacg cggttgcgcg gacggcttcc tgtgttttca ttggtcgaag cgttctgcgc
     4261 gtgcgcagag attccccagg gaaggcgggc cctcttccga atgtccaatc cggacattct
     4321 tccggttgga cctccgcggt cagcgctgtg cccggcgaga gatcacgtgg ggcgcggagg
     4381 cggtgctgct ggggcacggc cgtccagcct cggcggccat atttttgagg ggctgttcat
     4441 ctcgttcaca cgctctgtcc gccatgtttg tgagtggaag cgccattacc ccttcaagcg
     4501 actgaaggct gcagggcctc tggtggcccg catggggaga ccagacccgc caggcccgcc
     4561 tttccgcact cagtccgggc ttactttatt ttgtgagaca gggtctcgcc tagaggctcc
     4621 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     4681 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     4741 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     4801 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
     4861 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
     4921 acgcccctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
     4981 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggct
     5041 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
     5101 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
     5161 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
     5221 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
     5281 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
     5341 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
     5401 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
     5461 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     5521 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     5581 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     5641 gtttccccat actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     5701 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     5761 ggttcaaagt ttttttcttc catttaaggt gtcgtgaaaa ctaccccaag ctggcctctg
     5821 aggccaggga gacccaagct tatgtccgtc ctgacgccgc tgctgctgcg gggcttgaca
     5881 ggctcggccc ggcggctccc agtgccgcgc gccaagatcc attcgttggg ggatcccatg
     5941 tccgtcctga cgccgctgct gctgcggggc ttgacaggct cggcccggcg gctcccagtg
     6001 ccgcgcgcca agatccattc gttggggaag cttcgaattc tgcaggtcga cttcaagaac
     6061 ctgatctggc tgaaggaggt ggacagcacc caggagagac tgaaggagtg gaacgtgagc
     6121 tacggcaccg ccctggtggc cgacagacag accaggggca gaggcggctc gggcagaagg
     6181 tggtttagcc aggagggcgg cctgtacttc agcttcctgc tgaaccccaa ggagttcgag
     6241 aacctgctgc agctgcccct ggtgctgggc ctgagcgtga gcgaggccct ggaggagatc
     6301 accgagatcc ccttcagcct gaagtggccc aacgacgtgt acttccagga gagaaaggtg
     6361 agcggcgtgc tgtgcgagct gagcaaggac aagctgatcg tgggcatcgg catcaacgtg
     6421 aaccagagag agatccccga ggagatcaag gacagagcca ccaccctgta cgagatcacc
     6481 ggcaaggact gggacagaaa ggaggtgctg ctgaaggtgc tgaagagaat cagcgagaac
     6541 ctgaagaagt tcaaggaggc tagcgactac aaagacgatg acgacaaggg tacctaatag
     6601 tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     6661 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     6721 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     6781 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     6841 ggctctatgg cttctgaggc ggaaagaacc agctgggcct actagatagc ggacccctta
     6901 ccgaaacatc gccgcattct gcagaggagt cgaacttagt gtatgtaaac ttctgaccca
     6961 ctggaattgt gatacagtga attataagtg aaataatctg tctgtaaaca attgttggaa
     7021 aaatgacttg tgtcatgcac aaagtagatg tcctaactga cttgccaaaa ctattgtttg
     7081 ttaacaagaa atttgtggag tagttgaaaa acgagtttta atgactccaa cttaagtgta
     7141 tgtaaacttc cgacttcaac tgtataggga tcc
//
