LOCUS       sequence-433123-        4697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenously tag human 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 4697)
  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 4697)
  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-hEEA1 HDR template
FEATURES             Location/Qualifiers
     source          1..4697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     313..335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     327..344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     328..344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1084..1791
                     /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      1339..1348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1819..1875
                     /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             1876..1941
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2476..2492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2476..2492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2489..2511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2500..2516
                     /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(2524..2530,2531..2548,2549..2554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2569..2590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2707..2724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2878..3466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2958..2977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3637..4428,4429..4497))
                     /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     4260..4279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4498..4602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4670..4688
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
       61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      121 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      181 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      241 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      301 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
      361 cctcgcgaat gcatctagat acgctggagt ctgaggctcg tcctgaatga tatcaagctt
      421 gaattcgttt cgagtcagga gccatcgccg gaagggtcac accgacacat tttacaaaac
      481 agcctaaggc gtcccctcct cccgcctggg tcagggaggg acacccccag gggtcctcgc
      541 ccgcctccca ctagcgggca cgcggtcgtc gcgcgggcgc ggccctctga cggagcccag
      601 gcgtgcactc cgcagcctcg gcgtccccca gctagccgga agaagctgcg gcgacagcgt
      661 cctgccgcct gtctgtcacg cagccgccca cgttgccacc gcttccgatt ggtcgctggc
      721 ctggaccggc ccccttcagc cccgcctccc gcgaatggac aggcgagggg cgggcccgcc
      781 accgccccgc cccccggcgg ccgcgggcgg gcccgcccat ctgtcagtta cgggagctgc
      841 ccggcgggcg ggctggaggc gctcgcaggc tgcctagtcg ttcgctcgct ctctctcgcg
      901 tgctccctct cgccgcctgt ggggaagtcg gggccgcccg agcggccgtc gccaccgcct
      961 cgccccgacc tccttcccgg tcccctcccg gcccagtccc tcccgcccgc gcaccccgag
     1021 tagtgagtgg ccccgagcag ggtctggaga gtcaccgcgg cggcgccggg tggtggttaa
     1081 accatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta
     1141 cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat
     1201 ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc
     1261 ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac
     1321 gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt ccatcgcaca
     1381 atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc
     1441 cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg
     1501 accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa
     1561 accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc
     1621 actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag
     1681 ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag
     1741 gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa gtctagaagc
     1801 gctggtaccg gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa
     1861 gagaatcctg gaccggacta caaagaccat gacggtgatt ataaagatca tgacatcgat
     1921 tacaaggatg acgatgacaa gggcggatcc gggggaagcg gcggatcctt aaggaggatt
     1981 ttacagaggg taagagagtg aaaccgtctt tctggcagca cgtgagcggg acagccggag
     2041 ctcggggccg cggcgcgggc gcccctcctc agcgcgggct ccttccttcc ctaggcccca
     2101 cagcccggca ggcgcttagg cctcggtcgc ccggcccggg ggcggggagg gcgggtgggg
     2161 ccggcgcggc ggggcggcag gcagagccct ggtggccccg cgcgtccagg cctggcgccc
     2221 gctccggcgc ggggctccgt gtgtgcgagc gggctgcgag ctggagggtg tggtgccggc
     2281 gcgttcctcc cgggaaccct gagtgggagt gaaaatggag agttggtgcc cgtgggacgg
     2341 gccagccgcc gcctgccggg ctggagaaaa atcatacctg acctccatag cagaaagtca
     2401 aaagcctccg accggaggat cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag
     2461 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2521 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3061 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3121 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3181 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3241 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3301 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3361 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3421 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3481 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3541 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3601 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3661 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3721 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3781 tccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3841 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3901 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3961 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4021 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4081 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4141 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4201 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4261 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4321 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4381 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4441 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4501 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4561 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4621 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     4681 cacgaggccc tttcgtc
//
