LOCUS       sequence-341829-        8450 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 8450)
  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 8450)
  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..8450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2062..2179
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2231..2534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2535..2738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2689..2709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2735..2759
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2835..2844
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2844..2867
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2871..2894
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2898..2921
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    2937..2961
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3607..3631)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3644..4210
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3661..4212
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3805..3822)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4032..4051
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4224..4823
                     /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(4224..4243)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4720..4740
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4840..5428
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4893..4913)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5311..5322)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5503..5736
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5855..5873)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5941..6045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6046..6114,6115..6906)
                     /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(6264..6283)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7077..7665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7566..7585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7819..7836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8076..8097
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8112..8117,8118..8135,8136..8142)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8150..8166
                     /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     8155..8177
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8174..8190
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8174..8190
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8240..8259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttaattcaca
     2041 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2101 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2161 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggactag
     2221 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2281 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2341 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2401 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2461 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2521 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2581 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2641 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2701 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     2761 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccggcgg ccgcagatct
     2821 ctcgaggttg atctgccacc atggactaca aggatgacga tgacaagggc gattacaagg
     2881 atgacgacga taagggagat tacaaggatg acgacgataa ggctcgaggt tatcaaacaa
     2941 gtttgtacaa aaaagttggc atggctagtc gaggcgcaac aagacccaac gggccaaata
     3001 ctggaaataa aatatgccag ttcaaactag tacttctggg agagtccgct gttggcaaat
     3061 caagcctagt gcttcgtttt gtgaaaggcc aatttcatga atttcaagag agtaccattg
     3121 gggctgcttt tctaacccaa actgtatgtc ttgatgacac tacagtaaag tttgaaatat
     3181 gggatacagc tggtcaagaa cgataccata gcctagcacc aatgtactac agaggagcac
     3241 aagcagccat agttgtatat gatatcacaa atgaggagtc ctttgcaaga gcaaaaaatt
     3301 gggttaaaga acttcagagg caagcaagtc ctaacattgt aatagcttta tcgggaaaca
     3361 aggccgacct agcaaataaa agagcagtag atttccagga agcacagtcc tatgcagatg
     3421 acaatagttt attattcatg gagacatccg ctaaaacatc aatgaatgta aatgaaatat
     3481 tcatggcaat agctaaaaaa ttgccaaaga atgaaccaca aaatccagga gcaaattctg
     3541 ccagaggaag aggagtagac cttaccgaac ccacacaacc aaccaggaat cagtgttgta
     3601 gtaactaacc aactttcttg tacaaagtgg ttcgataacg aattccgccc ccccccccta
     3661 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     3721 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     3781 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     3841 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     3901 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     3961 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     4021 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4081 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4141 cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     4201 acgatgataa tatggccaca accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     4261 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     4321 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     4381 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     4441 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     4501 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     4561 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     4621 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     4681 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     4741 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     4801 tgacccgcaa gcccggtgcc tgatgcatcg atggatccta atcaacctct ggattacaaa
     4861 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     4921 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     4981 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     5041 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     5101 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     5161 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     5221 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     5281 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     5341 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     5401 cggatctccc tttgggccgc ctccccgcct gagatccttt aagaccaatg acttacaagg
     5461 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     5521 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     5581 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5641 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5701 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     5761 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggccc gggttaatta
     5821 aggaaagggc tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     5881 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5941 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6001 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6061 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6121 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6181 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6241 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     6301 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6361 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6421 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6481 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6541 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6601 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6661 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6721 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6781 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6841 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6901 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6961 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7021 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7081 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7141 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7201 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7261 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7321 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7381 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7441 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7501 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7561 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7621 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7681 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     7741 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     7801 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     7861 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa
     7921 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     7981 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt
     8041 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     8101 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     8161 taacaatttc acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt
     8221 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     8281 atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt
     8341 accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt
     8401 tcattttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt
//
