LOCUS       sequence-346179-        8452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For lentiviral transduction of 3FLAG-RAB5A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8452)
  AUTHORS   Park H, Hundley FV, Yu Q, Overmyer KA, Brademan DR, Serrano L, Paulo
            JA, Paoli JC, Swarup S, Coon JJ, Gygi SP, Wade Harper J
  TITLE     Spatial snapshots of amyloid precursor protein intramembrane 
            processing via early endosome proteomics.
  JOURNAL   Nat Commun. 2022 Oct 16;13(1):6112. doi: 10.1038/s41467-022-33881-x.
  PUBMED    36245040
REFERENCE   2  (bases 1 to 8452)
  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: "Nat 
            Commun."; date: "2022-10-16"; volume: "13(1):6112. doi"; pages: " 
            10.1038/s41467-022-33881-x"
COMMENT     Sequence Label: pHAGE-3FLAG-RAB5A
FEATURES             Location/Qualifiers
     source          1..8452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    47..172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    669..902
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1087..1131
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1428..1545
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        1597..1900
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1901..2104
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2055..2075
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2101..2125
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2201..2210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2210..2233
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2237..2260
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2264..2287
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    2303..2327
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2973..2997)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3010..3578
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3028..3580
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3172..3189)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3399..3418
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3592..4191
                     /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(3592..3611)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4088..4108
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4208..4796
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4261..4281)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4679..4690)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4871..5104
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5223..5241)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5309..5413
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5414..5482,5483..6274)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5632..5651)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6445..7033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6934..6953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7187..7204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7444..7465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7480..7485,7486..7503,7504..7510)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7518..7534
                     /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)."
     primer_bind     7523..7545
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7542..7558
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7542..7558
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7608..7627
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             7819..8452
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
       61 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      121 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      181 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      241 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      301 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
      361 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
      421 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
      481 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
      541 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
      601 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
      661 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
      721 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
      781 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
      841 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
      901 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
      961 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1021 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1081 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1141 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1201 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1261 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     1321 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct
     1381 cgacggtatc gccgaattaa ttcacaaatg gcagtattca tccacaattt taaaagaaaa
     1441 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     1501 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     1561 gacagcagag atccagtttg gactagtgga gttccgcgtt acataactta cggtaaatgg
     1621 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1681 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1741 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1801 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     1861 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     1921 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     1981 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2041 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2101 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
     2161 tagaagacac cggcggccgc agatctctcg aggttgatct gccaccatgg actacaagga
     2221 tgacgatgac aagggcgatt acaaggatga cgacgataag ggagattaca aggatgacga
     2281 cgataaggct cgaggttatc aaacaagttt gtacaaaaaa gttggcatgg ctagtcgagg
     2341 cgcaacaaga cccaacgggc caaatactgg aaataaaata tgccagttca aactagtact
     2401 tctgggagag tccgctgttg gcaaatcaag cctagtgctt cgttttgtga aaggccaatt
     2461 tcatgaattt caagagagta ccattggggc tgcttttcta acccaaactg tatgtcttga
     2521 tgacactaca gtaaagtttg aaatatggga tacagctggt caagaacgat accatagcct
     2581 agcaccaatg tactacagag gagcacaagc agccatagtt gtatatgata tcacaaatga
     2641 ggagtccttt gcaagagcaa aaaattgggt taaagaactt cagaggcaag caagtcctaa
     2701 cattgtaata gctttatcgg gaaacaaggc cgacctagca aataaaagag cagtagattt
     2761 ccaggaagca cagtcctatg cagatgacaa tagtttatta ttcatggaga catccgctaa
     2821 aacatcaatg aatgtaaatg aaatattcat ggcaatagct aaaaaattgc caaagaatga
     2881 accacaaaat ccaggagcaa attctgccag aggaagagga gtagacctta ccgaacccac
     2941 acaaccaacc aggaatcagt gttgtagtaa ctaaccaact ttcttgtaca aagtggttcg
     3001 ataacgaatt ccgccccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     3061 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     3121 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     3181 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     3241 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     3301 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     3361 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     3421 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3481 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3541 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catgaccgag
     3601 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     3661 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3721 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3781 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3841 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3901 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3961 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4021 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     4081 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4141 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg atgcatcgat
     4201 ggatcctaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4261 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4321 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4381 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4441 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4501 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4561 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     4621 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4681 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4741 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctga
     4801 gatcctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4861 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     4921 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4981 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     5041 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     5101 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     5161 tcagagagtg agaggcccgg gttaattaag gaaagggcta gatcattctt gaagacgaaa
     5221 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5281 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5341 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5401 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5461 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5521 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5581 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5641 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     5701 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5761 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5821 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5881 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5941 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6001 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6061 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6121 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6181 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6241 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6301 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6361 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6421 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6481 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6541 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6601 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6661 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6721 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6781 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6841 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6901 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6961 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7021 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7081 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7141 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7201 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     7261 ttaatgcagc aagctcatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     7321 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     7381 aaaaagctcc ccgtggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     7441 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     7501 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
     7561 gattacgaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     7621 actcatcaat gtatcttatc atgtctggat caactggata actcaagcta accaaaatca
     7681 tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt ttcatttact
     7741 ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg
     7801 tactacaaac ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg
     7861 atctgtggat ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc
     7921 caggggtcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag
     7981 ataaggtaga agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc
     8041 atgggatgga tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat
     8101 ttcatcacgt ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt
     8161 gctacaaggg actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg
     8221 agtggcgagc cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc
     8281 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     8341 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     8401 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag ca
//
