LOCUS       sequence-417658-        6435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the extracellular domain of human CD86 fused to 
            Spytag003 + Histag in HEK293 (or similar).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6435)
  AUTHORS   Patel A, Andre V, Eguiguren SB, Barton MI, Burton J, Denham EM, 
            Pettmann J, Morch AM, Kutuzov MA, Siller-Farfan JA, Dustin ML, van 
            der Merwe PA, Dushek O
  TITLE     Using CombiCells, a platform for titration and combinatorial display
            of cell surface ligands, to study T-cell antigen sensitivity 
            modulation by accessory receptors.
  JOURNAL   EMBO J. 2024 Jan;43(1):132-150. doi: 10.1038/s44318-023-00012-1. 
            Epub 2023 Dec 18.
  PUBMED    38177315
REFERENCE   2  (bases 1 to 6435)
  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: 
            "10.1038/s44318-023-00012-1"; journalName: "EMBO J"; date: 
            "2024-01"; volume: "43"; issue: "1"; pages: "132-150"
COMMENT     Sequence Label: pTT3-ecdCD86-ST3-His
FEATURES             Location/Qualifiers
     source          1..6435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(208..226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        294..398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(399..467,468..1259)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(617..636)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1430..2018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1919..1938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2172..2189
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2306..2327
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2342..2347,2348..2365,2366..2372)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2380..2396
                     /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)."
     primer_bind     2385..2407
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2404..2420
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2404..2420
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        2691..3070
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3071..3274
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3225..3245
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3271..3295
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     3776..3795
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3839..3848
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4583..4630
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             4631..4648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4678..4733
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4732..4751)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 catatgctat cctaatctat atctgggtag tatatgctat cctaatttat atctgggtag
       61 cataggctat cctaatctat atctgggtag catatgctat cctaatctat atctgggtag
      121 tatatgctat cctaatctgt atccgggtag catatgctat cctcatgata agctgtcaaa
      181 catgagaatt ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
      241 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      301 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      361 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      421 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      481 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      541 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      601 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
      661 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
      721 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
      781 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
      841 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
      901 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
      961 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1021 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1081 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1141 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1201 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1261 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1321 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1381 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1441 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1501 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1561 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1621 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1681 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1741 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1801 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1861 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1921 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1981 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2041 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2101 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2161 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2221 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2281 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     2341 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2401 acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc gaccaattct
     2461 catgtttgac agcttatcat cgcagatccg ggcaacgttg ttgccattgc tgcaggcgca
     2521 gaactggtag gtatggaaga tctatacatt gaatcaatat tggcaattag ccatattagt
     2581 cattggttat atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata
     2641 tcataatatg tacatttata ttggctcatg tccaatatga ccgccatgtt gacattgatt
     2701 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     2761 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     2821 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     2881 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     2941 tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     3001 ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     3061 tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc
     3121 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     3181 tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag
     3241 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcctcac
     3301 tctcttccgc atcgctgtct gcgagggcca gctgttgggc tcgcggttga ggacaaactc
     3361 ttcgcggtct ttccagtact cttggatcgg aaacccgtcg gcctccgaac ggtactccgc
     3421 caccgaggga cctgagcgag tccgcatcga ccggatcgga aaacctctcg agaaaggcgt
     3481 ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc gggcggcagc gggtggcggt
     3541 cggggttgtt tctggcggag gtgctgctga tgatgtaatt aaagtaggcg gtcttgagac
     3601 ggcggatggt cgaggtgagg tgtggcaggc ttgagatcca gctgttgggg tgagtactcc
     3661 ctctcaaaag cgggcattac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat
     3721 ttgatattca cctggcccga tctggccata cacttgagtg acaatgacat ccactttgcc
     3781 tttctctcca caggtgtcca ctcccaggtc caagtttaaa cggatctcta gcgaattcgc
     3841 caccatgggg atacttccca gccctgggat gcctgcgctg ctctccctcg tgagccttct
     3901 ctccgtgctg ctgatgggtt gcgtagctga aaccggtgct cctctgaaga ttcaagctta
     3961 tttcaatgag actgcagacc tgccatgcca atttgcaaac tctcaaaacc aaagcctgag
     4021 tgagctagta gtattttggc aggaccagga aaacttggtt ctgaatgagg tatacttagg
     4081 caaagagaaa tttgacagtg ttcattccaa gtatatgggc cgcacaagtt ttgattcgga
     4141 cagttggacc ctgagacttc acaatcttca gatcaaggac aagggcttgt atcaatgtat
     4201 catccatcac aaaaagccca caggaatgat tcgcatccac cagatgaatt ctgaactgtc
     4261 agtgcttgct aacttcagtc aacctgaaat agtaccaatt tctaatataa cagaaaatgt
     4321 gtacataaat ttgacctgct catctataca cggttaccca gaacctaaga agatgagtgt
     4381 tttgctaaga accaagaatt caactatcga gtatgatggt attatgcaga aatctcaaga
     4441 taatgtcaca gaactgtacg acgtttccat cagcttgtct gtttcattcc ctgatgttac
     4501 gagcaatatg accatcttct gtattctgga aactgacaag acgcggcttt tatcttcacc
     4561 tttctctata gagcttgagg acagaggcgt ccctcatatc gtgatggtcg acgcctataa
     4621 gaggtataaa catcatcacc atcaccatta aggatccccc gacctcgacc tctggctaat
     4681 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     4741 ggacatatgg gagggcaaat catttggtcg agatccctcg gagatctcta gctagaggat
     4801 cgatccccgc cccggacgaa ctaaacctga ctacgacatc tctgcccctt cttcgcgggg
     4861 cagtgcatgt aatcccttca gttggttggt acaacttgcc aactgggccc tgttccacat
     4921 gtgacacggg gggggaccaa acacaaaggg gttctctgac tgtagttgac atccttataa
     4981 atggatgtgc acatttgcca acactgagtg gctttcatcc tggagcagac tttgcagtct
     5041 gtggactgca acacaacatt gcctttatgt gtaactcttg gctgaagctc ttacaccaat
     5101 gctgggggac atgtacctcc caggggccca ggaagactac gggaggctac accaacgtca
     5161 atcagagggg cctgtgtagc taccgataag cggaccctca agagggcatt agcaatagtg
     5221 tttataaggc ccccttgtta accctaaacg ggtagcatat gcttcccggg tagtagtata
     5281 tactatccag actaacccta attcaatagc atatgttacc caacgggaag catatgctat
     5341 cgaattaggg ttagtaaaag ggtcctaagg aacagcgata tctcccaccc catgagctgt
     5401 cacggtttta tttacatggg gtcaggattc cacgagggta gtgaaccatt ttagtcacaa
     5461 gggcagtggc tgaagatcaa ggagcgggca gtgaactctc ctgaatcttc gcctgcttct
     5521 tcattctcct tcgtttagct aatagaataa ctgctgagtt gtgaacagta aggtgtatgt
     5581 gaggtgctcg aaaacaaggt ttcaggtgac gcccccagaa taaaatttgg acggggggtt
     5641 cagtggtggc attgtgctat gacaccaata taaccctcac aaaccccttg ggcaataaat
     5701 actagtgtag gaatgaaaca ttctgaatat ctttaacaat agaaatccat ggggtgggga
     5761 caagccgtaa agactggatg tccatctcac acgaatttat ggctatgggc aacacataat
     5821 cctagtgcaa tatgatactg gggttattaa gatgtgtccc aggcagggac caagacaggt
     5881 gaaccatgtt gttacactct atttgtaaca aggggaaaga gagtggacgc cgacagcagc
     5941 ggactccact ggttgtctct aacacccccg aaaattaaac ggggctccac gccaatgggg
     6001 cccataaaca aagacaagtg gccactcttt tttttgaaat tgtggagtgg gggcacgcgt
     6061 cagcccccac acgccgccct gcggttttgg actgtaaaat aagggtgtaa taacttggct
     6121 gattgtaacc ccgctaacca ctgcggtcaa accacttgcc cacaaaacca ctaatggcac
     6181 cccggggaat acctgcataa gtaggtgggc gggccaagat aggggcgcga ttgctgcgat
     6241 ctggaggaca aattacacac acttgcgcct gagcgccaag cacagggttg ttggtcctca
     6301 tattcacgag gtcgctgaga gcacggtggg ctaatgttgc catgggtagc atatactacc
     6361 caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatag gctatcctaa
     6421 tctatatctg ggtag
//
