LOCUS       40924_25711        6485 bp DNA     circular SYN 13-MAY-2021
DEFINITION  mammalian expression of CTLA4-mEos2.
ACCESSION   .
VERSION     .
KEYWORDS    .
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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6485)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2015-09-11"; pages: "
            10.1038/srep14072"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     promoter        639..657
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      709..718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1378..2055
                     /codon_start=1
                     /label=mEos2
                     /note="monomeric variant of green-to-red photoswitchable 
                     fluorescent protein EosFP (McKinney et al., 2009)"
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICIAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIHMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGYHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     intron          2128..2357
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     misc_feature    2419..2980
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             3013..3609
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     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"
     protein_bind    complement(4128..4144)
                     /label=lac operator
                     /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    complement(4197..4218)
                     /label=CAP binding site
                     /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"
     CDS             complement(5268..6125)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6126..6230)
                     /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
//
