LOCUS       sequence-444035-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of human CTLA4 with SNAP and 6xHis tags from 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  AUTHORS   Zhao Y, Lee CK, Lin CH, Gassen RB, Xu X, Huang Z, Xiao C, Bonorino 
            C, Lu LF, Bui JD, Hui E
  TITLE     PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28
            While Repressing the Inhibitory PD-1 and CTLA-4 Pathways.
  JOURNAL   Immunity. 2019 Dec 17;51(6):1059-1073.e9. doi: 
            10.1016/j.immuni.2019.11.003. Epub 2019 Nov 19.
  PUBMED    31757674
REFERENCE   2  (bases 1 to 7682)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.immuni.2019.11.003"; journalName: "Immunity"; date: 
            "2019-12-17- 17"; volume: "51"; issue: "6"; pages: "1059-1073.e9"
COMMENT     Sequence Label: pPPI4-SNAP-CTLA-4-His6
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(290..591)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     589..609
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(659..675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(659..675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(672..694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    683..699
                     /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(join(707..713,714..731,732..737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    752..773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(890..907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1061..1649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1141..1160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1820..2611,2612..2680))
                     /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     2443..2462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2681..2785)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2839..3218
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3219..3422
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3373..3393
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3419..3443
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          3558..4383
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             4477..5022
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             5041..5061
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             5446..5463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5520..5537)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5526..5750
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             6277..6840
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     polyA_signal    6911..7045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6948..6967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7002..7021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 aaagacagca ttttcaacaa atggtgctgg cacaactggt ggttatcatg gagaagaatg
       61 tgaattgatc cattccaatc tccttgtact aaggtcaaat ctaagtggat caaggaactc
      121 cacataaaac cagagacact gaaacttata gaggagaaag tggggaaaag cctcgaagat
      181 atgggcacag gggaaaaatt cctgaataga acagcaatgg cttgtgctgt aagatcgaga
      241 attgacaaat gggacctcat gaaactccaa agctatcgga tcaattcctc caaaaaagcc
      301 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
      361 aaaaattagt cagccatgca tggggcggag aatgggcgga actgggcgga gttaggggcg
      421 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
      481 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
      541 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
      601 acacattcca cagaattaat tcccgatccg tcgacctcga gagcttggcg taatcatggt
      661 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      721 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      781 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      841 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
      901 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
      961 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1021 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1081 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1141 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1201 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1261 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1321 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1381 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1441 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1501 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1561 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1621 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1681 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1741 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     1801 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     1861 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     1921 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     1981 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2041 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2101 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2161 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2221 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2281 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2341 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2401 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2461 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2521 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     2581 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2641 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2701 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2761 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     2821 gatcgggaga tccgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     2881 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     2941 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     3001 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     3061 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
     3121 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
     3181 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     3241 ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     3301 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc
     3361 ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     3421 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     3481 gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga acgcggattc
     3541 cccgtgccaa gagtgacgta agtaccgcct atagactcta taggcacacc cctttggctc
     3601 ttatgcatgc tatactgttt ttggcttggg gcctatacac ccccgcttcc ttatgctata
     3661 ggtgatggta tagcttagcc tataggtgtg ggttattgac cattattgac cactccccta
     3721 ttggtgacga tactttccat tactaatcca taacatggct ctttgccaca actatctcta
     3781 ttggctatat gccaatactc tgtccttcag agactgacac ggactctgta tttttacagg
     3841 atggggtccc atttattatt tacaaattca catatacaac aacgccgtcc cccgtgcccg
     3901 cagtttttat taaacatagc gtgggatctc cacgcgaatc tcgggtacgt gttccggaca
     3961 tgggctcttc tccggtagcg gcggagcttc cacatccgag ccctggtccc atgcctccag
     4021 cggctcatgg tcgctcggca gctccttgct cctaacagtg gaggccagac ttaggcacag
     4081 cacaatgccc accaccacca gtgtgccgca caaggccgtg gcggtagggt atgtgtctga
     4141 aaatgagctc ggagattggg ctcgcaccgc tgacgcagat ggaagactta aggcagcggc
     4201 agaagaagat gcaggcagct gagttgttgt attctgataa gagtcagagg taactcccgt
     4261 tgcggtgctg ttaacggtgg agggcagtgt agtctgagca gtactcgttg ctgccgcgcg
     4321 cgccaccaga cataatagct gacagactaa cagactgttc ctttccatgg gtcttttctg
     4381 cagatggacg ctatgaagag aggactgtgc tgcgtgctgc tgctgtgcgg agccgtgttc
     4441 gtgtcaccaa gccaggaaat ccatgctaga ttccgaatgg acaaagattg cgaaatgaaa
     4501 cgtaccaccc tggatagccc gctgggcaaa ctggaactga gcggctgcga acagggcctg
     4561 catgaaatta aactgctggg taaaggcacc agcgcggccg atgcggttga agttccggcc
     4621 ccggccgccg tgctgggtgg tccggaaccg ctgatgcagg cgaccgcgtg gctgaacgcg
     4681 tattttcatc agccggaagc gattgaagaa tttccggttc cggcgctgca tcatccggtg
     4741 tttcagcagg agagctttac ccgtcaggtg ctgtggaaac tgctgaaagt ggttaaattt
     4801 ggcgaagtga ttagctatca gcagctggcg gccctggcgg gtaatccggc ggccaccgcc
     4861 gccgttaaaa ccgcgctgag cggtaacccg gtgccgattc tgattccgtg ccatcgtgtg
     4921 gttagctcta gcggtgcggt tggcggttat gaaggtggtc tggcggtgaa agagtggctg
     4981 ctggcccatg aaggtcatcg tctgggtaaa ccgggtctgg gaggtagcgg tagcggtagc
     5041 gagactgtcc gattccaatc tgaaaaagca atgcacgtgg cccagcctgc tgtggtactg
     5101 gccagcagcc gaggcatcgc cagctttgtg tgtgagtatg catctccagg caaagccact
     5161 gaggtccggg tgacagtgct tcggcaggct gacagccagg tgactgaagt ctgtgcggca
     5221 acctacatga tggggaatga gttgaccttc ctagatgatt ccatctgcac gggcacctcc
     5281 agtggaaatc aagtgaacct cactatccaa ggactgaggg ccatggacac gggactctac
     5341 atctgcaagg tggagctcat gtacccaccg ccatactacc tgggcatagg caacggaacc
     5401 cagatttatg taattgatcc agaaccgtgc ccagattctg acggccatca ccatcaccat
     5461 cactgagcgg ccgctcgagc atgcatctag agcttgcgcc tctagagctc gctgatcagc
     5521 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     5581 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     5641 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     5701 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     5761 ggaaagaacc agctggggct cgagggggga tctcccgatc cccagctttg cttctcaatt
     5821 tcttatttgc ataatgagaa aaaaaggaaa attaatttta acaccaattc agtagttgat
     5881 tgagcaaatg cgttgccaaa aaggatgctt tagagacagt gttctctgca cagataagga
     5941 caaacattat tcagagggag tacccagagc tgagactcct aagccagtga gtggcacagc
     6001 atccagggag aaatatgctt gtcatcaccg aagcctgatt ccgtagagcc acaccctggt
     6061 aagggccaat ctgctcacac aggatagaga gggcaggagc cagggcagag catataaggt
     6121 gaggtaggat cagttgctcc tcacatttgc ttctgacata gttgtgttgg gagcttggat
     6181 agcttggggg ggggacagct cagggctgcg atttcgcgcc aaacttgacg gcaatcctag
     6241 cgtgaaggct ggtaggattt tatccccgct gccatcatgg ttcgaccatt gaactgcatc
     6301 gtcgccgtgt cccaaaatat ggggattggc aagaacggag acctaccctg gcctccgctc
     6361 aggaacgagt tcaagtactt ccaaagaatg accacaacct cttcagtgga aggtaaacag
     6421 aatctggtga ttatgggtag gaaaacctgg ttctccattc ctgagaagaa tcgaccttta
     6481 aaggacagaa ttaatatagt tctcagtaga gaactcaaag aaccaccacg aggagctcat
     6541 tttcttgcca aaagtttgga tgatgcctta agacttattg aacaaccgga attggcaagt
     6601 aaagtagaca tggtttggat agtcggaggc agttctgttt accaggaagc catgaatcaa
     6661 ccaggccacc tcagactctt tgtgacaagg atcatgcagg aatttgaaag tgacacgttt
     6721 ttcccagaaa ttgatttggg gaaatataaa cttctcccag aatacccagg cgtcctctct
     6781 gaggtccagg aggaaaaagg catcaagtat aagtttgaag tctacgagaa gaaagactaa
     6841 caggaagatg ctttcaagtt ctctgctccc ctcctaaagc tatgcatttt tataagacca
     6901 tgctagcttg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6961 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7021 caatgtatct tatcatgtct ggatcaacga tagcttatct gtgggcgatg ccaagcacct
     7081 ggatgctgtt ggtttcctgc tactgattta gaagccattt gccccctgag tggggcttgg
     7141 gagcactaac tttctctttc aaaggaagca atgcagaaag aaaagcatac aaagtataag
     7201 ctgccatgta ataatggaag aagataaggt tgtatgaatt agatttacat acttctgaat
     7261 tgaaactaaa cacctttaaa ttcttaaata tataacacat ttcatatgaa agtattttac
     7321 ataagtaact cagatacata gaaaacaaag ctaatgatag gtgtccctaa aagttcattt
     7381 attaattcta caaatgatga gctggccatc aaaattccag ctcaattctt caacgaatta
     7441 gaaagagcaa tctgcaaact catctggaat aacaaaaaac ctaggatagc aaaaactctt
     7501 ctcaaggata aaagaacctc tggtggaatc accatgcctg acctaaagct gtactacaga
     7561 gcaattgtga taaaaactgc atggtactga tatagaaacg gacaagtaga ccaatggaat
     7621 agaacccaca cacctatggt cacttgatct tcaacaagag agctaaaacc atccactgga
     7681 aa
//
