LOCUS Exported 9105 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses V5-tagged Sec61B in lentiviral vector. ACCESSION . VERSION . KEYWORDS pLV-EF1a-V5-Sec61B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9105) TITLE Gerace lab plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 9105) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9105 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..116 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1 (lacking the first T)" promoter 194..405 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" intron 406..1344 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1301..1321 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1360..1369 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1369..1410 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 1432..1719 /codon_start=1 /gene="SEC61B" /product="human Sec61-beta subunit of the translocon in the endoplasmic reticulum" /label=Sec61-beta /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS" primer_bind complement(2016..2033) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2243..2262 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2424..3023 /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(2424..2443) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2920..2940 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3047..3635 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3100..3120) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3518..3529) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3842..4475 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(4603..4619) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4603..4619) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4616..4638) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4627..4643 /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(4651..4681) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4696..4717 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4834..4851) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5005..5593) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5085..5104) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5764..6624) /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 6387..6406 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6625..6729) /gene="bla" /label=AmpR promoter polyA_signal 6777..6911 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6814..6833) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6868..6887 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 7079..7712 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 7759..7884 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8381..8614 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8799..8843 /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" ORIGIN 1 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata 61 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg 121 gtttattaca gggacagcag agatccagtt tatcgatgag gccctttcgt cttcactcga 181 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg 241 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc 301 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc 361 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt 421 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc 481 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt 541 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg 601 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat 661 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat 721 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc 781 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg 841 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc 901 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg 961 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg 1021 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca 1081 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc 1141 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt 1201 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc 1261 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt 1321 caaagttttt ttcttccatt tcaggtgtcg tgaggatccg ccgccatggg taagcctatc 1381 cctaaccctc tcctcggtct cgattctacg gggggcggag ggggtgggcc catgcctggt 1441 ccgaccccca gtggcactaa cgtgggatcc tcagggcgct ctcccagcaa agcagtggcc 1501 gcccgggcgg cgggatccac tgtccggcag aggaaaaatg ccagctgtgg gacaaggagt 1561 gcaggccgca caacctcggc aggcaccggg gggatgtggc gattctacac agaagattca 1621 cctgggctca aagttggccc tgttccagta ttggttatga gtcttctgtt catcgcttct 1681 gtatttatgt tgcacatttg gggcaagtac actcgttcgt ataatgggcc gcgacgatgt 1741 ttaaacttcg aaacgcgtga attcctcgag ggcggccgct ctagagtcga cgggccgcgg 1801 taacaattgt taactaactt aagctagcaa cggtttccct ctagcgggat caattccgcc 1861 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta 1921 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc 1981 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct 2041 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt 2101 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa 2161 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg 2221 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga 2281 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac 2341 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt 2401 cctttgaaaa acacgataat accatgaccg agtacaagcc cacggtgcgc ctcgccaccc 2461 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 2521 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct 2581 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 2641 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 2701 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 2761 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 2821 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 2881 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 2941 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca 3001 tgacccgcaa gcccggtgcc tgacccgggc ggggcgcgtc tggaacaatc aacctctgga 3061 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 3121 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 3181 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 3241 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 3301 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 3361 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 3421 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac 3481 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 3541 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 3601 gacgagtcgg atctcccttt gggccgcctc cccgcctgga attaattctg cagtcgagac 3661 ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc 3721 tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta 3781 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 3841 ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 3901 acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 3961 ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 4021 aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 4081 gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 4141 gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 4201 gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 4261 tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 4321 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 4381 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 4441 cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 4501 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 4561 gcgtttaccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 4621 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 4681 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 4741 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 4801 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 4861 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4921 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4981 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 5041 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 5101 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 5161 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 5221 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 5281 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5341 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 5401 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 5461 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 5521 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5581 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5641 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5701 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5761 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5821 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 5881 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5941 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 6001 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 6061 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 6121 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 6181 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6241 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6301 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6361 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6421 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6481 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6541 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6601 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6661 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6721 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcaact 6781 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 6841 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 6901 atgtctggat caactggata actcaagcta accaaaatca tcccaaactt cccaccccat 6961 accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg 7021 aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac ttagtagttg 7081 gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat ctaccacaca 7141 caaggctact tccctgatta gcagaactac acaccagggc caggggtcag atatccactg 7201 acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga agaggccaat 7261 aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga tgacccggag 7321 agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt ggcccgagag 7381 ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg actttccgct 7441 ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc cctcagatcc 7501 tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc 7561 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg 7621 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag 7681 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg 7741 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca 7801 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag 7861 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa 7921 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca 7981 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt 8041 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 8101 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc 8161 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa 8221 gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 8281 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 8341 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 8401 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 8461 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 8521 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 8581 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 8641 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 8701 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 8761 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 8821 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 8881 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 8941 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 9001 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 9061 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgcc //