LOCUS Exported 7724 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION NFAT-driven ZsGreen-1 reporter gene with mutated human CD4 gene. ACCESSION . VERSION . KEYWORDS 8xNFAT-ZsG-muhCD4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7724) TITLE Nakayama lab plasmids REFERENCE 2 (bases 1 to 7724) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7724 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 26..405 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 406..609 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 560..580 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 610..785 /label=5' LTR (truncated) /note="truncated long terminal repeat from Moloney murine sarcoma virus" misc_feature 848..1047 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 1248..1664 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1594..1616 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1632..1648 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" protein_bind 1698..1727 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1728..1757 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1758..1787 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1788..1817 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1818..1847 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1848..1877 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1878..1907 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" protein_bind 1908..1937 /label=NFAT binding site /bound_moiety="NFAT transcription factor" /note="NFAT binding site from the human interleukin 2 (IL-2) gene (Mattila et al., 1990)" promoter 1949..1980 /label=minP /note="minimal TATA-box promoter with low basal activity" regulatory 1994..2003 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2000..2695 /codon_start=1 /product="Zoanthus green fluorescent protein" /label=ZsGreen1 /note="mammalian codon-optimized" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH AIASGSALP" promoter 2718..3217 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2753..2776) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3150..3169 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3235..4611 /codon_start=1 /product="human CD4 cell surface glycoprotein" /label=CD4 /translation="MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQ KKSIQFHWKNSNQIKILGNYGSFLWKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDS DTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNI QGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFS FPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLH LTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLML SLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMAL IVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI " LTR 4692..5117 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus" rep_origin complement(5287..5875) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5367..5386) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6046..6906) /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 6669..6688 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6907..7011) /gene="bla" /label=AmpR promoter primer_bind 7079..7097 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7135..7157) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 7257..7276 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7470..7492 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7620..7639 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 gatatcccaa cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca 61 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 121 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 181 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 241 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 301 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 361 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 421 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 481 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 541 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 601 agcagagctc aataaaagag cccacaaccc ctcactcggc gcgccagtct tccgatagac 661 tgcgtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat ccgaatcgtg 721 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccacga cgggggtctt 781 tcatttgggg gctcgtccgg gatttggaga cccctgccca gggaccaccg acccaccacc 841 gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg tctatgtttg 901 atgttatgcg cctgcgtctg tactagttag ctaactagct ctgtatctgg cggacccgtg 961 gtggaactga cgagttctga acacccggcc gcaaccctgg gagacgtccc agggactttg 1021 ggggccgttt ttgtggcccg acctgaggaa gggagtcgat gtggaatccg accccgtcag 1081 gatatgtggt tctggtagga gacgagaacc taaaacagtt cccgcctccg tctgaatttt 1141 tgctttcggt ttggaaccga agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc 1201 tgtgttgtct ctgtctgact gtgtttctgt atttgtctga aaattagggc cagactgtta 1261 ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc gctcacaacc 1321 agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa tggccaacct 1381 ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc caggttaaga 1441 tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac atcgtgacct 1501 gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac cctaagcctc 1561 cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt tcgaccccgc 1621 ctcgatcctc cctttatcca gccctcactc cttctctagg cgccggaatt gaagatcatc 1681 atgtctggat ccaagctgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg 1741 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg 1801 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg 1861 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg 1921 tttcatacag aaggcgtcgc ggagactcta gagggtatat aatggaagct cgaattccag 1981 tccccgagat ctcgccacca tggcccagtc caagcacggc ctgaccaagg agatgaccat 2041 gaagtaccgc atggagggct gcgtggacgg ccacaagttc gtgatcaccg gcgagggcat 2101 cggctacccc ttcaagggca agcaggccat caacctgtgc gtggtggagg gcggcccctt 2161 gcccttcgcc gaggacatct tgtccgccgc cttcatgtac ggcaaccgcg tgttcaccga 2221 gtacccccag gacatcgtcg actacttcaa gaactcctgc cccgccggct acacctggga 2281 ccgctccttc ctgttcgagg acggcgccgt gtgcatctgc aacgccgaca tcaccgtgag 2341 cgtggaggag aactgcatgt accacgagtc caagttctac ggcgtgaact tccccgccga 2401 cggccccgtg atgaagaaga tgaccgacaa ctgggagccc tcctgcgaga agatcatccc 2461 cgtgcccaag cagggcatct tgaagggcga cgtgagcatg tacctgctgc tgaaggacgg 2521 tggccgcttg cgctgccagt tcgacaccgt gtacaaggcc aagtccgtgc cccgcaagat 2581 gcccgactgg cacttcatcc agcacaagct gacccgcgag gaccgcagcg acgccaagaa 2641 ccagaagtgg cacctgaccg agcacgccat cgcctccggc tccgccttgc cctgactcga 2701 ggttaacgaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc 2761 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc 2821 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc 2881 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg 2941 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg 3001 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc 3061 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc 3121 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc 3181 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatcccgc caccatgaac 3241 cggggagtcc cttttaggca cttgcttctg gtgctgcaac tggcgctcct cccagcagcc 3301 actcagggaa agaaagtggt gctgggcaaa aaaggggata cagtggaact gacctgtaca 3361 gcttcccaga agaagagcat acaattccac tggaaaaact ccaaccagat aaagattctg 3421 ggaaattacg gctccttctt atggaaaggt ccatccaagc tgaatgatcg cgctgactca 3481 agaagaagcc tttgggacca aggaaacttc cccctgatca tcaagaatct taagatagaa 3541 gactcagata cttacatctg tgaagtggag gaccagaagg aggaggtgca attgctagtg 3601 ttcggattga ctgccaactc tgacacccac ctgcttcagg ggcagagcct gaccctgacc 3661 ttggagagcc cccctggtag tagcccctca gtgcaatgta ggagtccaag gggtaaaaac 3721 atacaggggg ggaagaccct ctccgtgtct cagctggagc tccaggatag tggcacctgg 3781 acatgcactg tcttgcagaa ccagaagaag gtggagttca aaatagacat cgtggtgcta 3841 gctttccaga aggcctccag catagtctat aagaaagagg gggaacaggt ggagttctcc 3901 ttcccactcg cctttacagt tgaaaagctg acgggcagtg gcgagctgtg gtggcaggcg 3961 gagagggctt cctcctccaa gtcttggatc acctttgacc tgaagaacaa ggaagtgtct 4021 gtaaaacggg ttacccagga ccctaagctc cagatgggca agaagctccc gctccacctc 4081 accctgcccc aggccttgcc tcagtatgct ggctctggaa acctcaccct ggcccttgaa 4141 gcgaaaacag gaaagttgca ccaggaagtg aacctggtgg tgatgagagc cactcagctc 4201 cagaaaaatt tgacctgtga ggtgtgggga cccacctccc ctaagctgat gctgagcttg 4261 aaactggaga acaaggaggc aaaggtctcg aagcgggaga aggcggtgtg ggtgctgaac 4321 cctgaggcgg ggatgtggca gtgtctgctg agtgactcgg gacaggtcct gctggaatcc 4381 aacatcaagg ttctgcccac atggtcgacc ccggtgcagc caatggccct gattgtgctg 4441 gggggcgtcg ccggcctcct gctgttcatt gggctaggca tcttcttctg tgtcaggtgc 4501 cggcaccgaa ggcgccaagc agagcggatg tctcagatca agagactcct cagtgagaag 4561 aagacctgcc agtgccctca ccggtttcag aagacatgta gccccatttg aaagcttcac 4621 cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag 4681 aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca 4741 ttttgcaagg catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga 4801 acagatggaa cagggtcgac cctagagaac catcagatgt ttccagggtg ccccaaggac 4861 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg 4921 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt 4981 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca 5041 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc 5101 agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac 5161 cgacccacca ccgggaggta agctggctgc ctcgcgcgtt tcggtgatga cggtgaaaac 5221 ctctgacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5701 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 7021 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 7081 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 7141 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 7201 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 7261 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 7321 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg 7381 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 7441 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggcgca 7501 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc 7561 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg 7621 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg 7681 atgcgtccgg cgtagaggcg attagtccaa tttgttaaag acag //