LOCUS       sequence-443131-        6749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the extracellular domain of human CD80 fused to 
            Spytag003 + Histag in HEK293 (or similar).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6749)
  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 6749)
  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-ecdCD80-ST3-His
FEATURES             Location/Qualifiers
     source          1..6749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     734..758
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1239..1258
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1302..1311
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2034..2081
                     /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             2082..2099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2129..2184
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2183..2202)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4420..4438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4506..4610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4611..4679,4680..5471)
                     /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(4829..4848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5642..6230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6131..6150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6384..6401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6518..6539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6554..6559,6560..6577,6578..6584)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6592..6608
                     /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     6597..6619
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6616..6632
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6616..6632
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agatctatac attgaatcaa tattggcaat tagccatatt agtcattggt tatatagcat
       61 aaatcaatat tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt
      121 atattggctc atgtccaata tgaccgccat gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctcgt ttagtgaacc gtcagatcct cactctcttc cgcatcgctg
      781 tctgcgaggg ccagctgttg ggctcgcggt tgaggacaaa ctcttcgcgg tctttccagt
      841 actcttggat cggaaacccg tcggcctccg aacggtactc cgccaccgag ggacctgagc
      901 gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg cgtctaacca gtcacagtcg
      961 caaggtaggc tgagcaccgt ggcgggcggc agcgggtggc ggtcggggtt gtttctggcg
     1021 gaggtgctgc tgatgatgta attaaagtag gcggtcttga gacggcggat ggtcgaggtg
     1081 aggtgtggca ggcttgagat ccagctgttg gggtgagtac tccctctcaa aagcgggcat
     1141 tacttctgcg ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc
     1201 cgatctggcc atacacttga gtgacaatga catccacttt gcctttctct ccacaggtgt
     1261 ccactcccag gtccaagttt aaacggatct ctagcgaatt cgccaccatg ggacacacaa
     1321 gaaggcaggg cacaagcccc agcaagtgcc cctacctgaa cttcttccag ctgctggtgc
     1381 tggccggcct gagccatttt tgttctggcg tgatccacgt gaccaaagaa gtgaaagagg
     1441 tcgccacact gagctgcggc cacaatgtgt ccgtggaaga actggcccag accagaatct
     1501 actggcagaa agaaaagaaa atggtgctga ccatgatgag cggcgacatg aacatctggc
     1561 ccgagtacaa gaaccggacc atcttcgaca tcaccaacaa cctgagcatc gtgatcctgg
     1621 ctctgaggcc ttctgacgag ggcacctatg agtgcgtggt gctgaagtac gagaaggacg
     1681 ccttcaagcg ggaacacctg gccgaagtga cactgagcgt gaaggccgac tttcccacac
     1741 ctagcatcag cgacttcgag atccccacca gcaacatccg gcggatcatc tgttctacca
     1801 gcggcggctt tcctgagcct cacctgagct ggcttgagaa cggcgaggaa ctgaacgcca
     1861 tcaacaccac cgtgtctcag gaccccgaga cagagctgta tgccgtgtcc agcaagctgg
     1921 acttcaacat gaccaccaac cacagcttca tgtgcctgat taagtacggc cacctgagag
     1981 tgaaccagac cttcaactgg aataccacca agcaagagca cttccccgac aacagaggcg
     2041 tccctcatat cgtgatggtc gacgcctata agaggtataa acatcatcat catcaccatt
     2101 aaggatcccc cgacctcgac ctctggctaa taaaggaaat ttattttcat tgcaatagtg
     2161 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttggtc
     2221 gagatccctc ggagatctct agctagagga tcgatccccg ccccggacga actaaacctg
     2281 actacgacat ctctgcccct tcttcgcggg gcagtgcatg taatcccttc agttggttgg
     2341 tacaacttgc caactgggcc ctgttccaca tgtgacacgg ggggggacca aacacaaagg
     2401 ggttctctga ctgtagttga catccttata aatggatgtg cacatttgcc aacactgagt
     2461 ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat tgcctttatg
     2521 tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc ccaggggccc
     2581 aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag ctaccgataa
     2641 gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt aaccctaaac
     2701 gggtagcata tgcttcccgg gtagtagtat atactatcca gactaaccct aattcaatag
     2761 catatgttac ccaacgggaa gcatatgcta tcgaattagg gttagtaaaa gggtcctaag
     2821 gaacagcgat atctcccacc ccatgagctg tcacggtttt atttacatgg ggtcaggatt
     2881 ccacgagggt agtgaaccat tttagtcaca agggcagtgg ctgaagatca aggagcgggc
     2941 agtgaactct cctgaatctt cgcctgcttc ttcattctcc ttcgtttagc taatagaata
     3001 actgctgagt tgtgaacagt aaggtgtatg tgaggtgctc gaaaacaagg tttcaggtga
     3061 cgcccccaga ataaaatttg gacggggggt tcagtggtgg cattgtgcta tgacaccaat
     3121 ataaccctca caaacccctt gggcaataaa tactagtgta ggaatgaaac attctgaata
     3181 tctttaacaa tagaaatcca tggggtgggg acaagccgta aagactggat gtccatctca
     3241 cacgaattta tggctatggg caacacataa tcctagtgca atatgatact ggggttatta
     3301 agatgtgtcc caggcaggga ccaagacagg tgaaccatgt tgttacactc tatttgtaac
     3361 aaggggaaag agagtggacg ccgacagcag cggactccac tggttgtctc taacaccccc
     3421 gaaaattaaa cggggctcca cgccaatggg gcccataaac aaagacaagt ggccactctt
     3481 ttttttgaaa ttgtggagtg ggggcacgcg tcagccccca cacgccgccc tgcggttttg
     3541 gactgtaaaa taagggtgta ataacttggc tgattgtaac cccgctaacc actgcggtca
     3601 aaccacttgc ccacaaaacc actaatggca ccccggggaa tacctgcata agtaggtggg
     3661 cgggccaaga taggggcgcg attgctgcga tctggaggac aaattacaca cacttgcgcc
     3721 tgagcgccaa gcacagggtt gttggtcctc atattcacga ggtcgctgag agcacggtgg
     3781 gctaatgttg ccatgggtag catatactac ccaaatatct ggatagcata tgctatccta
     3841 atctatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta
     3901 atctatatct gggtagtata tgctatccta atttatatct gggtagcata ggctatccta
     3961 atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta
     4021 atctgtatcc gggtagcata tgctacccag atagagatta gggtagtata tgctatccta
     4081 atttatatct gggtagcata tactacccaa atatctggat agcatatgct atcctaatct
     4141 atatctgggt agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
     4201 atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaattt
     4261 atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct
     4321 atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct atcctcatga
     4381 taagctgtca aacatgagaa ttttcttgaa gacgaaaggg cctcgtgata cgcctatttt
     4441 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4501 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4561 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4621 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4681 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4741 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4801 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     4861 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4921 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4981 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5041 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5101 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     5161 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5221 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5281 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5341 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5401 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5461 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5521 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5581 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5641 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5701 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5761 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5821 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5881 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5941 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6001 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6061 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6121 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6181 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6241 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6301 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6361 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6421 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6481 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6541 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6601 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc tagctagagg
     6661 tcgaccaatt ctcatgtttg acagcttatc atcgcagatc cgggcaacgt tgttgccatt
     6721 gctgcaggcg cagaactggt aggtatgga
//
