LOCUS Exported 8486 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PresentER with removable cassette for cloning of MHC-I antigen (GFP version). ACCESSION . VERSION . KEYWORDS PresentER-Cassette GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8486) AUTHORS Gejman RS, Chang AY, Jones HF, DiKun K, Hakimi AA, Schietinger A, Scheinberg DA TITLE Rejection of immunogenic tumor clones is limited by clonal fraction. JOURNAL Elife. 2018 Nov 30;7. pii: 41090. doi: 10.7554/eLife.41090. PUBMED 30499773 REFERENCE 2 (bases 1 to 8486) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8486 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 29..445 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 375..397 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 413..429 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" ncRNA 485..578 /label=5' miR-30a /note="sequence upstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" ncRNA 1184..1296 /label=3' miR-30a /note="sequence downstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" promoter 1310..1809 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(1345..1368) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 1742..1761 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 1830..2429 /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(1830..1849) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2326..2346 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 2506..3057 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2650..2667) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2877..2896 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3069..3788 /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 3069..3785 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3114..3135) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3375..3394) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3722..3743 /label=EGFP-C /note="EGFP, forward primer" LTR 3890..4404 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" primer_bind complement(4573..4589) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4573..4589) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4586..4608) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4597..4613 /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(4621..4651) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4666..4687 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4804..4821) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4975..5563) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5055..5074) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5734..6594) /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 6357..6376 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6595..6699) /gene="bla" /label=AmpR promoter primer_bind 6767..6785 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6823..6845) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6945..6964 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7158..7180 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7308..7327 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 7527..8043 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" misc_feature 8107..8448 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" ORIGIN 1 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac 61 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa 121 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg 181 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg 241 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga 301 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc 361 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc 421 agccctcact ccttctctag gcgccggaat tagatctctc gactagggat aacagggtaa 481 ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt 541 gctgggatta cttcttcagg ttaacccaac agaaggctcg aggtgactgg agttcagacg 601 tgtgctcttc cgatcgccac catgcctaat catcagtccg ggtcacctac cggcagttca 661 gacctgctcc ttgatggcaa gaaacaacga gcccatctgg cgctgaggag aaaacggcga 721 cgggaaatgc gcaagattaa ccgaaaggtg agaagaatga atctcgcacc cattaaagaa 781 aaaacagcct ggcagcacct gcaagctctg atcttcgagg cggaagaagt gttgaagact 841 tctcaaactc cacagacctc cctcacgctg tttctagcac tcttggccgt attggccaca 901 tttgcttctg acacaactgt gttcactagc aacctcaaac agacaccatg gtgcatctga 961 ctcctgagga gaagtctgcc gttactgccc tgtggggcaa ggtgaacgtg gatgaagttg 1021 gtggtgaggc cctgggcagg ttggtatcaa ggttacaaga caggtttaag gagaccaata 1081 gaaactgggc atgtggagac agagaagggc caaacaggcc aaagatcgga agagcgtcgt 1141 gtagggaaag agtgtagatc tcggtggtcg ccgtatcatt gaattcaagg ggctacttta 1201 ggagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat acatttttac 1261 aaagctgaat taaaatggta taaattaaat cacttttttc aattctaccg ggtaggggag 1321 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc 1381 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct 1441 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc 1501 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt 1561 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc 1621 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg 1681 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg 1741 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg 1801 ggcctttcga cctgcagccc aagcttacca tgaccgagta caagcccacg gtgcgcctcg 1861 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc 1921 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag 1981 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg 2041 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga 2101 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag 2161 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct 2221 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg 2281 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct 2341 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct 2401 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg 2461 aaaggagcgc acgaccccat catccaattc cgcccccccc ccctaacgtt actggccgaa 2521 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt 2581 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg 2641 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc 2701 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc 2761 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa 2821 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc 2881 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg 2941 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg 3001 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg 3061 ccacaaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 3121 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 3181 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 3241 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 3301 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 3361 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 3421 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 3481 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 3541 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 3601 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 3661 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 3721 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 3781 acaagtaatg aattaattaa gaattatcaa gcttatcgat accgtcgacc tgcagccaag 3841 cttatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc 3901 cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaataca 3961 taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca gaatatgggc 4021 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc 4081 cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc 4141 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct 4201 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc 4261 gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt gtatccaata aaccctcttg 4321 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac 4381 tacccgtcag cgggggtctt tcatgggtaa cagtttcttg aagttggaga acaacattct 4441 gagggtagga gtcgaatatt aagtaatcct gactcaatta gccactgttt tgaatccaca 4501 tactccaata ctcctgaaat agttcattat ggacagcgca gaagagctgg ggagaattaa 4561 ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4621 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4681 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4801 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4861 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4921 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4981 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 5041 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 5101 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 5161 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 5221 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 5281 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 5341 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5401 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5461 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5521 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5581 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5641 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5701 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5761 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5821 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5881 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5941 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6001 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6061 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6121 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6181 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6241 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6301 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6361 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6421 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6481 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6541 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6601 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6661 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6721 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 6781 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 6841 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 6901 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat 6961 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 7021 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 7081 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 7141 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca 7201 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 7261 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg 7321 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg 7381 tagaggcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg 7441 actcaacaat atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt 7501 ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta 7561 gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca 7621 gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag 7681 cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca 7741 gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg 7801 tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc 7861 ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg 7921 cgtcgcccgg gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtgga 7981 ctcgctgatc cttgggaggg tctcctcaga ttgattgact gcccacctcg ggggtctttc 8041 atttggaggt tccaccgaga tttggagacc cctgcccagg gaccaccgac ccccccgccg 8101 ggaggtaagc tggccagcgg tcgtttcgtg tctgtctctg tctttgtgcg tgtttgtgcc 8161 ggcatctaat gtttgcgcct gcgtctgtac tagttagcta actagctctg tatctggcgg 8221 acccgtggtg gaactgacga gttctgaaca cccggccgca accctgggag acgtcccagg 8281 gactttgggg gccgtttttg tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc 8341 ccgtcaggat atgtggttct ggtaggagac gagaacctaa aacagttccc gcctccgtct 8401 gaatttttgc tttcggtttg gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc 8461 atcgttctgt gttgtctctg tctgac //