LOCUS Exported 8450 bp ds-DNA circular SYN 16-DEC-2021 DEFINITION For lentiviral transduction of 3FLAG-RAB5A. ACCESSION . VERSION . KEYWORDS pHAGE-3FLAG-RAB5A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8450) AUTHORS Harper lab TITLE HOLD - Early Endosome Proteomics and its Application to Spatial Snapshots of Amyloid Precursor Protein Intramembrane Processing JOURNAL unpublished REFERENCE 2 (bases 1 to 8450) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 16, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8450 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 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 6046..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" primer_bind 7928..7947 /label=SV40pro-F /note="SV40 promoter/origin, 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 8112..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 //