LOCUS       sequence-429350-        4465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenously tag mouse EEA1 with an N-terminal 3xFLAG in addition to
            cytosolic mNG for cell sorting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4465)
  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 4465)
  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: mNeonGreen-P2A-3xFLAG-msEEA1 HDR template
FEATURES             Location/Qualifiers
     source          1..4465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(27..131)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     199..217
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(255..277)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     377..396
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     590..612
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     604..621
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     605..621
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1092..1799
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1347..1356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1827..1883
                     /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             1884..1949
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2470..2486)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2470..2486)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2483..2505)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2494..2510
                     /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)."
     promoter        complement(join(2518..2524,2525..2542,2543..2548))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2563..2584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2701..2718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2872..3460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2952..2971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3631..4422,4423..4465,1..26))
                     /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     4254..4273
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
       61 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
      121 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
      181 atgacattaa cctataaaaa taggcgtatc acgaggccct tttgtctcgc gcgtttcggt
      241 gatgacggtg aaaacctctg acacatgcag ctcccggaga ctgtcacagc ttgtctgtaa
      301 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
      361 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
      421 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
      481 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
      541 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
      601 cgttgtaaaa cgacggccag tgaattgacg cgtattggga tcaagctctc ggcggaagaa
      661 gccaaggcac cagcttcctg tggcctgtct ctcacactag ccccgcttcc gattggtcga
      721 agcctcgggc cggccctccc tccggccccg ccctccccgc ggatggacag gccgaggagg
      781 cggggctgca gcggctccgc ccctcggccc cgggcgggcg ggcccgccca gctgtcagtt
      841 acgggggcgg cccggcaggc gggcgctcgc tggctccctc ctcgctggct cgtgctcgtg
      901 agcctctcgc cgcctgtggg gaagtcgggg ccgccaaagc cgcgtccacc gccgcctctt
      961 ccccgccctc ctccctcccg gctcggctgc cggcccctcg cgccctccag tcgtgagtaa
     1021 cccgcataag ggcgcggaga gtccccgcgg cggcggcggc ggtgacggcg gcggcggcgg
     1081 ccggccaaac catggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     1141 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     1201 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     1261 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     1321 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccgga taccaagtcc
     1381 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     1441 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     1501 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     1561 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     1621 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1681 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1741 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     1801 ctagaagcgc tggtaccgga tccggcgcaa caaacttctc tctgctgaaa caagccggag
     1861 atgtcgaaga gaatcctgga ccggactaca aagaccatga cggtgattat aaagatcatg
     1921 acatcgatta caaggatgac gatgacaagg gcggatccgg gggaagcggc ggatcctttc
     1981 gaagaatctt gcagagggta agagtgaaac cgtccttccg gcagcacgtg agcgcgagcg
     2041 tccgagcccg gccgcggggc agcccgggct ggagggcggg ggcgctcaga gctccgctag
     2101 gcccggtgac agcgtgcgcc gaggcctgct ggtcctgccg gagccgggct gcgggtgggg
     2161 cggggcgcag gccggagccc ggagggcctc cagcctcggc tcccgcgctc ggggctgtca
     2221 ccacgggctg gagggtgtgg tgcccgcgcg cgcccctccc cgctccccgc tcccggctcc
     2281 gcgcccgtgg gtgctcggct gcgggcggct gcggggccct tccctgcccc agcgtgccac
     2341 caccttccca cgacacgcac ctgcctgtct gcctccttac tcatctatct gcacggtaca
     2401 ggagggtcat gggagacccg gcctagtcct gggacatccc aatggcgcgc cgagcttggc
     2461 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2521 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2581 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2641 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct gttccgcttc
     2701 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2761 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2821 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2881 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2941 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3001 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3061 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3121 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3181 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3241 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3301 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3361 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3421 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3481 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3541 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3601 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3661 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3721 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagaaccacg
     3781 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3841 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3901 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3961 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4021 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4081 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4141 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4201 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4261 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4321 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4381 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4441 aaatgccgca aaaaagggaa taagg
//
