LOCUS       sequence-429344-        4071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenously tag mouse TMEM115 with a C-terminal 3xHA tag in 
            addition to cytosolic mScarlet-I for cell sorting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4071)
  AUTHORS   Hollingsworth LR, Veeraraghavan P, Paulo JA, Harper JW
  TITLE     Spatiotemporal proteomic profiling of cellular responses to NLRP3 
            agonists
  JOURNAL   bioRxiv 2024.04.19.590338
REFERENCE   2  (bases 1 to 4071)
  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: "bioRxiv 
            2024.04.19.590338"
COMMENT     Sequence Label: pTwist Amp msTMEM115-TEV-3C-P2A-mScarlet-I HDR...
FEATURES             Location/Qualifiers
     source          1..4071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2..697
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(135..153)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     403..422
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1223..1239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1223..1239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1236..1258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1411..1999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1491..1510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(2116..2140)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(2240..3031,3032..3100))
                     /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     2863..2882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3101..3205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3304..3326
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3318..3335
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3319..3335
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3905..3925
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3926..4006
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             join(4016..4071,1)
                     /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"
ORIGIN
        1 gatggtgagc aagggcgagg cagtgatcaa ggagttcatg cggttcaagg tgcacatgga
       61 gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      121 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tctcctggga
      181 catcctgtcc cctcagttca tgtacggctc cagggccttc atcaagcacc ccgccgacat
      241 ccccgactac tataagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      301 cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc ctggaggacg gcaccctgat
      361 ctacaaggtg aagctccgcg gcaccaactt ccctcctgac ggccccgtaa tgcagaagaa
      421 gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc gaggacggcg tgctgaaggg
      481 cgacattaag atggccctgc gcctgaagga cggcggccgc tacctggcgg acttcaagac
      541 cacctacaag gccaagaagc ccgtgcagat gcccggcgcc tacaacgtcg accgcaagtt
      601 ggacatcacc tcccacaacg aggactacac cgtggtggaa cagtacgaac gctccgaggg
      661 ccgccactcc accggcggca tggacgagct gtacaagtag accactttga gtgcagttct
      721 tctactccca tgccttcccg atccgtctcg gctactgcag agcactgact gctctgagga
      781 gagggaagag ggtctgctgg gggttcctgt ggccttccgc tgtgtctgga caacactaac
      841 acaggaccct tgttgctacc tggttctgac tccagacaac cacaatgcca ggtacggggt
      901 ctctgagcag cagccagtgc agatccccat ttgcagtaag attgtacctc agcggtacaa
      961 ttcctcacca aggaagtggc tgcagggaca ctggtgccac ggctcctggg ccagccctta
     1021 tgtctgaaac tggttgccaa cagccccgag ccaaggcaag gatttgtgtt tgccaaaaat
     1081 gttctgggga cccagccagt gtgggtctga acatccccgc ccacccccag actgcattct
     1141 catcagggtt tcttgtgccc ttctgcaggc taggtggagg ctcagtgatg ataagtctgc
     1201 gatggtggat gcatgtgtca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1261 gagggcacaa tcctattccg cgctatccga caatctccaa gacattaggt ggagttcagt
     1321 tcggcgtatg gcatatgtcg ctggaaagaa catgtgagca aaaggccagc aaaaggccag
     1381 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1441 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1501 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1561 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     1621 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1681 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1741 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1801 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1861 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1921 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1981 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctctatt
     2041 caacaaagcc gccgtcccgt caagtcagcg taaatgggta gggggcttca aatcgtcctc
     2101 gtgataccaa ttcggagcct gcttttttgt acaaacttgt tgataatggc aattcaagga
     2161 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2221 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2281 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2341 agggcttacc atctggcccc agtgctgcaa tgataccgcg agagccacgc tcaccggctc
     2401 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2461 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2521 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2581 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2641 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2701 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2761 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2821 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2881 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2941 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3001 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3061 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3121 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3181 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccagat acctgaaaca
     3241 aaacccatcg tacggccaag gaagtctcca ataactgtga tccaccacaa gcgccagggt
     3301 tttcccagtc acgacgttgt aaaacgacgg ccagtcatgc ataatccgca cgcatctgga
     3361 ataaggaagt gccattccgc ctgacctact gaaacacgcc actcctgaca gaagcactac
     3421 acccacccgt ctttagctag caaactcaga agtatccgtg gtggctggcc tgctaggtag
     3481 gctgccaaca tctaggtagc cttctcagtc tggaggtgcc ttgggtcctc attcaattgc
     3541 caaagcagat cctggcccag gctcttagac gcggcacggg gatgggaaac gtgggatttg
     3601 gaggcaaacc tctttggtag gaggatttgg ccttattgaa gaaggttgca gagtatgttg
     3661 gtctcccaaa cccagtgtaa cctgcctttt ctcttctggc tccaggcaac tagccctaaa
     3721 ggctctcaat gagcggctga agagagtgga ggatcagtca gcctggccca gcatggatga
     3781 tgacgaagag gaagctgggg caaaaacgga cagtcctctg cccttagaag aagcttccac
     3841 gccacccgga aaagtgacag tgccggaatc cagtctcatc accttggaga cagctcccct
     3901 actcgaaaac ctgtacttcc agggctaccc atacgatgtt ccagattacg cttatcctta
     3961 tgacgtacct gactatgcat acccttatga tgtaccagac tacgctggat ccggcgcaac
     4021 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac c
//
