LOCUS       sequence-417657-        7149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the extracellular domain of human ICAM1 fused to 
            Spytag003 + Histag in HEK293 (or similar).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7149)
  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 7149)
  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-ecdICAM1-ST3-His
FEATURES             Location/Qualifiers
     source          1..7149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(199..217)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        285..389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(390..458,459..1250)
                     /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(608..627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1421..2009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1910..1929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2163..2180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2297..2318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2333..2338,2339..2356,2357..2363)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2371..2387
                     /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     2376..2398
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2395..2411
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2395..2411
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        2682..3061
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3062..3265
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3216..3236
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3262..3286
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     3767..3786
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3830..3839
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5288..5335
                     /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             5336..5353
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    5383..5438
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5437..5456)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 tcctaatcta tatctgggta gtatatgcta tcctaattta tatctgggta gcataggcta
       61 tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta
      121 tcctaatctg tatccgggta gcatatgcta tcctcatgat aagctgtcaa acatgagaat
      181 tttcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
      241 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      361 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      421 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      481 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      541 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      601 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
      661 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      721 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
      781 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      841 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      901 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
      961 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1021 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1081 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1141 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1201 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1261 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1321 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1381 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1441 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1501 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1561 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2041 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2101 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2161 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     2221 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2281 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     2341 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     2401 acagctatga ccatgattac gccaagctct agctagaggt cgaccaattc tcatgtttga
     2461 cagcttatca tcgcagatcc gggcaacgtt gttgccattg ctgcaggcgc agaactggta
     2521 ggtatggaag atctatacat tgaatcaata ttggcaatta gccatattag tcattggtta
     2581 tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat
     2641 gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag
     2701 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     2761 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     2821 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     2881 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     2941 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     3001 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     3061 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     3121 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     3181 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     3241 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca ctctcttccg
     3301 catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact cttcgcggtc
     3361 tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg
     3421 acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt
     3481 cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt
     3541 ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga cggcggatgg
     3601 tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc cctctcaaaa
     3661 gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc
     3721 acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
     3781 acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattcg ccaccatggg
     3841 gatacttccc agccctggga tgcctgcgct gctctccctc gtgagccttc tctccgtgct
     3901 gctgatgggt tgcgtagctg aaaccggtca gacatctgtg tccccctcaa aagtcatcct
     3961 gccccgggga ggctccgtgc tggtgacatg cagcacctcc tgtgaccagc ccaagttgtt
     4021 gggcatagag accccgttgc ctaaaaagga gttgctcctg cctgggaaca accggaaggt
     4081 gtatgaactg agcaatgtgc aagaagatag ccaaccaatg tgctattcaa actgccctga
     4141 tgggcagtca acagctaaaa ccttcctcac cgtgtactgg actccagaac gggtggaact
     4201 ggcacccctc ccctcttggc agccagtggg caagaacctt accctacgct gccaggtgga
     4261 gggtggggca ccccgggcca acctcaccgt ggtgctgctc cgtggggaga aggagctgaa
     4321 acgggagcca gctgtggggg agcccgctga ggtcacgacc acggtgctgg tgaggagaga
     4381 tcaccatgga gccaatttct cgtgccgcac tgaactggac ctgcggcccc aagggctgga
     4441 gctgtttgag aacacctcgg ccccctacca gctccagacc tttgtcctgc cagcgactcc
     4501 cccacaactt gtcagccccc gggtcctaga ggtggacacg caggggaccg tggtctgttc
     4561 cctggacagg ctgttcccag tctcggaggc ccaggtccac ctggcactgg gggaccagag
     4621 gttgaacccc acagtcacct atggcaacga ctccttctcg gccaaggcct cagtcagtgt
     4681 gaccgcagag gacgagggca cccagcggct gacgtgtgca gtaatactgg ggaaccagag
     4741 ccaggagaca ctgcagacag tgaccatcta cagctttccg gcgcccaacg tgattctgac
     4801 gaagccagag gtctcagaag ggaccgaggt gacagtgaag tgtgaggccc accctagagc
     4861 caaggtgacg ctgaatgggg ttccagccca gccactgggc ccgagggccc agctcctgct
     4921 gaaggccacc ccagaggaca acgggcgcag cttctcctgc tctgcaaccc tggaggtggc
     4981 cggccagctt atacacaaga accagacccg ggagcttcgt gtcctgtatg gcccccgact
     5041 ggacgagagg gattgtccgg gaaactggac gtggccagaa aattcccagc agactccaat
     5101 gtgccaggct tgggggaacc cattgcccga gctcaagtgt ctaaaggatg gcactttccc
     5161 actgcccatc ggggaatcag tgactgtcac tcgagatctt gagggcacct acctctgtcg
     5221 ggccaggagc actcaagggg aggtcacccg cgaggtgacc gtgaatgtgc tctccccccg
     5281 gtatgagaga ggcgtccctc atatcgtgat ggtcgacgcc tataagaggt ataaacatca
     5341 tcaccatcac cattaaggat cccccgacct cgacctctgg ctaataaagg aaatttattt
     5401 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     5461 caaatcattt ggtcgagatc cctcggagat ctctagctag aggatcgatc cccgccccgg
     5521 acgaactaaa cctgactacg acatctctgc cccttcttcg cggggcagtg catgtaatcc
     5581 cttcagttgg ttggtacaac ttgccaactg ggccctgttc cacatgtgac acgggggggg
     5641 accaaacaca aaggggttct ctgactgtag ttgacatcct tataaatgga tgtgcacatt
     5701 tgccaacact gagtggcttt catcctggag cagactttgc agtctgtgga ctgcaacaca
     5761 acattgcctt tatgtgtaac tcttggctga agctcttaca ccaatgctgg gggacatgta
     5821 cctcccaggg gcccaggaag actacgggag gctacaccaa cgtcaatcag aggggcctgt
     5881 gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat aaggccccct
     5941 tgttaaccct aaacgggtag catatgcttc ccgggtagta gtatatacta tccagactaa
     6001 ccctaattca atagcatatg ttacccaacg ggaagcatat gctatcgaat tagggttagt
     6061 aaaagggtcc taaggaacag cgatatctcc caccccatga gctgtcacgg ttttatttac
     6121 atggggtcag gattccacga gggtagtgaa ccattttagt cacaagggca gtggctgaag
     6181 atcaaggagc gggcagtgaa ctctcctgaa tcttcgcctg cttcttcatt ctccttcgtt
     6241 tagctaatag aataactgct gagttgtgaa cagtaaggtg tatgtgaggt gctcgaaaac
     6301 aaggtttcag gtgacgcccc cagaataaaa tttggacggg gggttcagtg gtggcattgt
     6361 gctatgacac caatataacc ctcacaaacc ccttgggcaa taaatactag tgtaggaatg
     6421 aaacattctg aatatcttta acaatagaaa tccatggggt ggggacaagc cgtaaagact
     6481 ggatgtccat ctcacacgaa tttatggcta tgggcaacac ataatcctag tgcaatatga
     6541 tactggggtt attaagatgt gtcccaggca gggaccaaga caggtgaacc atgttgttac
     6601 actctatttg taacaagggg aaagagagtg gacgccgaca gcagcggact ccactggttg
     6661 tctctaacac ccccgaaaat taaacggggc tccacgccaa tggggcccat aaacaaagac
     6721 aagtggccac tctttttttt gaaattgtgg agtgggggca cgcgtcagcc cccacacgcc
     6781 gccctgcggt tttggactgt aaaataaggg tgtaataact tggctgattg taaccccgct
     6841 aaccactgcg gtcaaaccac ttgcccacaa aaccactaat ggcaccccgg ggaatacctg
     6901 cataagtagg tgggcgggcc aagatagggg cgcgattgct gcgatctgga ggacaaatta
     6961 cacacacttg cgcctgagcg ccaagcacag ggttgttggt cctcatattc acgaggtcgc
     7021 tgagagcacg gtgggctaat gttgccatgg gtagcatata ctacccaaat atctggatag
     7081 catatgctat cctaatctat atctgggtag cataggctat cctaatctat atctgggtag
     7141 catatgcta
//
