LOCUS       sequence-444032-        7904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of human CTLA4 with Twin-Strep, SNAP, GCN4 and 
            6xHis tags from mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7904)
  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 7904)
  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-Strep-SNAP-CTLA-4-GCN4-His6
FEATURES             Location/Qualifiers
     source          1..7904
                     /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             join(4477..4500,4501..4536,4537..4560)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4561..4584
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4585..5130
                     /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             5149..5169
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             5596..5652
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             5668..5685
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5742..5759)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5748..5972
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             6499..7062
                     /codon_start=1
                     /gene="Mus musculus Dhfr"
                     /product="mouse dihydrofolate reductase"
                     /label=DHFR
                     /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     polyA_signal    7133..7267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7170..7189)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7224..7243
                     /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 ttccgatgga gccacccgca gttcgagaaa
     4501 ggtggaggtt ccggaggtgg atcgggaggt agtgcttggt ctcatcctca attcgagaag
     4561 ctggaagttc tgttccaggg gcccatggac aaagattgcg aaatgaaacg taccaccctg
     4621 gatagcccgc tgggcaaact ggaactgagc ggctgcgaac agggcctgca tgaaattaaa
     4681 ctgctgggta aaggcaccag cgcggccgat gcggttgaag ttccggcccc ggccgccgtg
     4741 ctgggtggtc cggaaccgct gatgcaggcg accgcgtggc tgaacgcgta ttttcatcag
     4801 ccggaagcga ttgaagaatt tccggttccg gcgctgcatc atccggtgtt tcagcaggag
     4861 agctttaccc gtcaggtgct gtggaaactg ctgaaagtgg ttaaatttgg cgaagtgatt
     4921 agctatcagc agctggcggc cctggcgggt aatccggcgg ccaccgccgc cgttaaaacc
     4981 gcgctgagcg gtaacccggt gccgattctg attccgtgcc atcgtgtggt tagctctagc
     5041 ggtgcggttg gcggttatga aggtggtctg gcggtgaaag agtggctgct ggcccatgaa
     5101 ggtcatcgtc tgggtaaacc gggtctggga ggtagcggta gcggtagcga gactgtccga
     5161 ttccaatctg aaaaagcaat gcacgtggcc cagcctgctg tggtactggc cagcagccga
     5221 ggcatcgcca gctttgtgtg tgagtatgca tctccaggca aagccactga ggtccgggtg
     5281 acagtgcttc ggcaggctga cagccaggtg actgaagtct gtgcggcaac ctacatgatg
     5341 gggaatgagt tgaccttcct agatgattcc atctgcacgg gcacctccag tggaaatcaa
     5401 gtgaacctca ctatccaagg actgagggcc atggacacgg gactctacat ctgcaaggtg
     5461 gagctcatgt acccaccgcc atactacctg ggcataggca acggaaccca gatttatgta
     5521 attgatccag aaccgtgccc agattctgac ttcggtggtt ctggtggttc tatgaagcaa
     5581 ctggaggata aggtagagga acttctgtcc aagaactacc atcttgaaaa tgaggtggcc
     5641 cgcttgaaaa agcttgtggg cgagagacat caccatcacc atcactgagc ggccgctcga
     5701 gcatgcatct agagcttgcg cctctagagc tcgctgatca gcctcgactg tgccttctag
     5761 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     5821 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     5881 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5941 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     6001 ctcgaggggg gatctcccga tccccagctt tgcttctcaa tttcttattt gcataatgag
     6061 aaaaaaagga aaattaattt taacaccaat tcagtagttg attgagcaaa tgcgttgcca
     6121 aaaaggatgc tttagagaca gtgttctctg cacagataag gacaaacatt attcagaggg
     6181 agtacccaga gctgagactc ctaagccagt gagtggcaca gcatccaggg agaaatatgc
     6241 ttgtcatcac cgaagcctga ttccgtagag ccacaccctg gtaagggcca atctgctcac
     6301 acaggataga gagggcagga gccagggcag agcatataag gtgaggtagg atcagttgct
     6361 cctcacattt gcttctgaca tagttgtgtt gggagcttgg atagcttggg ggggggacag
     6421 ctcagggctg cgatttcgcg ccaaacttga cggcaatcct agcgtgaagg ctggtaggat
     6481 tttatccccg ctgccatcat ggttcgacca ttgaactgca tcgtcgccgt gtcccaaaat
     6541 atggggattg gcaagaacgg agacctaccc tggcctccgc tcaggaacga gttcaagtac
     6601 ttccaaagaa tgaccacaac ctcttcagtg gaaggtaaac agaatctggt gattatgggt
     6661 aggaaaacct ggttctccat tcctgagaag aatcgacctt taaaggacag aattaatata
     6721 gttctcagta gagaactcaa agaaccacca cgaggagctc attttcttgc caaaagtttg
     6781 gatgatgcct taagacttat tgaacaaccg gaattggcaa gtaaagtaga catggtttgg
     6841 atagtcggag gcagttctgt ttaccaggaa gccatgaatc aaccaggcca cctcagactc
     6901 tttgtgacaa ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg
     6961 gggaaatata aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa
     7021 ggcatcaagt ataagtttga agtctacgag aagaaagact aacaggaaga tgctttcaag
     7081 ttctctgctc ccctcctaaa gctatgcatt tttataagac catgctagct tgaacttgtt
     7141 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     7201 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     7261 ctggatcaac gatagcttat ctgtgggcga tgccaagcac ctggatgctg ttggtttcct
     7321 gctactgatt tagaagccat ttgccccctg agtggggctt gggagcacta actttctctt
     7381 tcaaaggaag caatgcagaa agaaaagcat acaaagtata agctgccatg taataatgga
     7441 agaagataag gttgtatgaa ttagatttac atacttctga attgaaacta aacaccttta
     7501 aattcttaaa tatataacac atttcatatg aaagtatttt acataagtaa ctcagataca
     7561 tagaaaacaa agctaatgat aggtgtccct aaaagttcat ttattaattc tacaaatgat
     7621 gagctggcca tcaaaattcc agctcaattc ttcaacgaat tagaaagagc aatctgcaaa
     7681 ctcatctgga ataacaaaaa acctaggata gcaaaaactc ttctcaagga taaaagaacc
     7741 tctggtggaa tcaccatgcc tgacctaaag ctgtactaca gagcaattgt gataaaaact
     7801 gcatggtact gatatagaaa cggacaagta gaccaatgga atagaaccca cacacctatg
     7861 gtcacttgat cttcaacaag agagctaaaa ccatccactg gaaa
//
