LOCUS Exported 6485 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression of CTLA4-mEos2. ACCESSION . VERSION . KEYWORDS pirespuro2-CTLA4-mEos2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6485) AUTHORS Fricke F, Beaudouin J, Eils R, Heilemann M TITLE One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy. JOURNAL Sci Rep. 2015 Sep 11;5:14072. doi: 10.1038/srep14072. PUBMED 26358640 REFERENCE 2 (bases 1 to 6485) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6485 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 11..390 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 391..594 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 545..565 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 639..658 /label=T7 /note="T7 promoter, forward primer" promoter 639..657 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 709..718 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" intron 2128..2357 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 2340..2359 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 2518..2980 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2574..2591) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2801..2820 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3013..3612 /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(3013..3032) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3509..3529 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(3748..3765) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3754..3978 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 4025..4044 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4104..4120) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4104..4120) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4117..4139) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4128..4144 /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(4152..4182) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4197..4218 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4335..4352) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4506..5094) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4586..4605) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5265..6125) /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 5888..5907 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6126..6230) /gene="bla" /label=AmpR promoter primer_bind complement(6305..6324) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 361 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 421 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 481 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 541 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 601 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 661 acccaagctt ggtaccgagc tcggatcgat atctgcggcc tagctagcgc caccatggct 721 tgccttggat ttcagcggca caaggctcag ctgaacctgg ctaccaggac ctggccctgc 781 actctcctgt tttttcttct cttcatccct gtcttctgca aagcaatgca cgtggcccag 841 cctgctgtgg tactggccag cagccgaggc atcgccagct ttgtgtgtga gtatgcatct 901 ccaggcaaag ccactgaggt ccgggtgaca gtgcttcggc aggctgacag ccaggtgact 961 gaagtctgtg cggcaaccta catgatgggg aatgagttga ccttcctaga tgattccatc 1021 tgcacgggca cctccagtgg aaatcaagtg aacctcacta tccaaggact gagggccatg 1081 gacacgggac tctacatctg caaggtggag ctcatgtacc caccgccata ctacctgggc 1141 ataggcaacg gaacccagat ttatgtaatt gatccagaac cgtgcccaga ttctgacttc 1201 ctcctctgga tccttgcagc agttagttcg gggttgtttt tttatagctt tctcctcaca 1261 gctgtttctt tgagcaaaat gctaaagaaa agaagccctc ttacaacagg ggtcggaggt 1321 ggaccggtgc cccagtggga gggattcgct gcgctgctgg caaccccggt cgctactatg 1381 tctgctatca agccagacat gaagatcaag ttgagaatgg aaggtaacgt taacggtcac 1441 cacttcgtta tcgacggtga cggtactggt aagccattcg aaggtaagca atctatggac 1501 ttggaagtta aggaaggtgg tccattgcca ttcgctttcg acatcttgac tactgctttc 1561 cactacggta acagagtttt cgctaagtac ccagacaaca tccaagacta cttcaagcaa 1621 tctttcccaa agggttactc ttgggaaaga tctttgactt tcgaagacgg tggtatctgt 1681 atcgctagaa acgacatcac tatggaaggt gacactttct acaacaaggt tagattctac 1741 ggtactaact tcccagctaa cggtccagtt atgcaaaaga agactttgaa gtgggaacca 1801 tctactgaaa agatgtacgt tagagacggt gttttgactg gtgacatcca catggctttg 1861 ttgttggaag gtaacgctca ctacagatgt gacttcagaa ctacttacaa ggctaaggaa 1921 aagggtgtta agttgccagg ttaccacttc gttgaccact gtatcgaaat cttgtctcac 1981 gacaaggact acaacaaggt taagttgtac gaacacgctg ttgctcactc tggtttgcca 2041 gacaacgcta gaagataaag cggccgcata gataactgat ccagtgtgct ggaattaatt 2101 cgctgtctgc gagggccagc tgttggggtg agtactccct ctcaaaagcg ggcatgactt 2161 ctgcgctaag attgtcagtt tccaaaaacg aggaggattt gatattcacc tggcccgcgg 2221 tgatgccttt gagggtggcc gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa 2281 gcttgaggtg tggcaggctt gagatctggc catacacttg agtgacaatg acatccactt 2341 tgcctttctc tccacaggtg tccactccca ggtccaactg caggtcgagc atgcatctag 2401 ggcggccaat tccgcccctc nnncccctaa cgttactggc cgaagccgct tggaataagg 2461 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag 2521 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 2581 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 2641 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 2701 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 2761 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 2821 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 2881 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa 2941 ccacggggac gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccacaagg 3001 agacgacctt ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 3061 ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 3121 gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 3181 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 3241 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 3301 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 3361 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 3421 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 3481 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 3541 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 3601 cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc 3661 catggctccg accgaagcca cccggggcgg ccccgccgac cccgcacccg cccccgaggc 3721 ccaccgactc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 3781 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 3841 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 3901 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3961 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg agtgcattct 4021 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4081 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4141 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4201 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4321 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4381 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4441 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4501 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4561 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4621 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4681 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4741 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4801 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4861 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4921 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4981 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5041 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5101 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5161 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5221 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5281 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5341 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5401 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5461 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5521 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5581 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 5641 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5701 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5761 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 5821 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 5881 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 5941 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6001 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6061 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6121 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6181 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6241 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 6301 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 6361 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 6421 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 6481 ccaga //