LOCUS Exported 6474 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION 3rd gen lentiviral eGFP expression vector, CMV promoter, Zeo. ACCESSION . VERSION . KEYWORDS pLenti CMV GFP Zeo (637-7) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6474) AUTHORS Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK, Kaufman PD. TITLE A versatile viral system for expression and depletion of proteins in mammalian cells. JOURNAL PLoS One. 2009 Aug 6;4(8):e6529. PUBMED 19657394 REFERENCE 2 (bases 1 to 6474) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6474 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 71..659 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 560..579 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 813..830 /label=L4440 /note="L4440 vector, forward primer" protein_bind 947..968 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 983..1013 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1021..1037 /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 1026..1048 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1045..1061 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1045..1061 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 1080..1100 /label=T3 /note="T3 promoter, forward primer" promoter 1082..1100 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 1128..1354 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 1355..1535 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1582..1707 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 2200..2433 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2618..2662 /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 2929..3045 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" enhancer 3068..3371 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3372..3575 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 3526..3546 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 3572..3596 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 3665..3674 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3671..4390 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3671..4387 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3716..3737) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3977..3996) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4324..4345 /label=EGFP-C /note="EGFP, forward primer" misc_feature 4406..4994 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4459..4479) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4877..4888) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5045..5065) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5050..5379 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5230..5365 /label=SV40 ori /note="SV40 origin of replication" promoter 5391..5438 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5457..5831 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" LTR 5924..6157 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 6229..6350 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6266..6285) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6320..6339 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 61 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 121 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 181 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 241 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 301 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 361 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 421 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 481 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 541 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 601 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 661 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 721 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 781 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 841 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 901 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 961 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 1021 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg 1081 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc 1141 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga 1201 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg 1261 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga 1321 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg 1381 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 1441 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 1501 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa 1561 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg 1621 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag 1681 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg 1741 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg 1801 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc 1861 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga 1921 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac 1981 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag 2041 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga 2101 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa 2161 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt 2221 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag 2281 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca 2341 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc 2401 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact 2461 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat 2521 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat 2581 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga 2641 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat 2701 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact 2761 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc 2821 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag 2881 agacagatcc attcgattag tgaacggatc tcgacggtat cggttaactt ttaaaagaaa 2941 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat 3001 acaaactaaa gaattacaaa aacaaattac aaaattcaaa attttatcga taagcttggg 3061 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 3121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 3181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 3241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 3301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 3361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 3421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 3481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 3541 ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct 3601 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgactctag aggatccacc 3661 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt 3721 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga 3781 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc 3841 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga 3901 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 3961 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 4021 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat 4081 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa 4141 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt 4201 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc 4261 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga 4321 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct 4381 gtacaagtaa agcggccgcg tcgacaatca acctctggat tacaaaattt gtgaaagatt 4441 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 4501 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 4561 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 4621 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 4681 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 4741 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 4801 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 4861 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 4921 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 4981 ggccgcctcc ccgcctggaa ttctgcagat atccggttag taatgagttt ggaattaatt 5041 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 5101 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 5161 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 5221 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 5281 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 5341 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccct gttgacaatt 5401 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg 5461 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt 5521 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg 5581 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca 5641 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg 5701 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt 5761 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg 5821 agcaggactg acacgtgcta cgagatttaa atggtacctt taagaccaat gacttacaag 5881 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctagctcac 5941 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc 6001 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 6061 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 6121 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt 6181 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt 6241 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 6301 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg 6361 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 6421 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccg //