LOCUS       sequence-443124-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the extracellular domain of human CD19 fused to 
            Spytag003 + Histag in HEK293(or similar) with SUMO for better 
            protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  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 6873)
  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-ecdCD19-ST3-His_withSUMO
FEATURES             Location/Qualifiers
     source          1..6873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        215..594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        595..798
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     749..769
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     795..819
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1300..1319
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1363..1372
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1453..1740
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1747..1770
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2545..2592
                     /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             2593..2610
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2640..2695
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2694..2713)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4605..4623)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4691..4795
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4796..4864,4865..5656)
                     /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(5014..5033)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5827..6415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6316..6335
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6569..6586
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6703..6724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6739..6744,6745..6762,6763..6769)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6777..6793
                     /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     6782..6804
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6801..6817
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6801..6817
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tcatcgcaga tccgggcaac gttgttgcca ttgctgcagg cgcagaactg gtaggtatgg
       61 aagatctata cattgaatca atattggcaa ttagccatat tagtcattgg ttatatagca
      121 taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt
      181 tatattggct catgtccaat atgaccgcca tgttgacatt gattattgac tagttattaa
      241 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      301 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      361 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      421 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc
      481 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      541 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      601 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      661 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      721 aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag
      781 gtctatataa gcagagctcg tttagtgaac cgtcagatcc tcactctctt ccgcatcgct
      841 gtctgcgagg gccagctgtt gggctcgcgg ttgaggacaa actcttcgcg gtctttccag
      901 tactcttgga tcggaaaccc gtcggcctcc gaacggtact ccgccaccga gggacctgag
      961 cgagtccgca tcgaccggat cggaaaacct ctcgagaaag gcgtctaacc agtcacagtc
     1021 gcaaggtagg ctgagcaccg tggcgggcgg cagcgggtgg cggtcggggt tgtttctggc
     1081 ggaggtgctg ctgatgatgt aattaaagta ggcggtcttg agacggcgga tggtcgaggt
     1141 gaggtgtggc aggcttgaga tccagctgtt ggggtgagta ctccctctca aaagcgggca
     1201 ttacttctgc gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc
     1261 ccgatctggc catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg
     1321 tccactccca ggtccaagtt taaacggatc tctagcgaat tcgccaccat ggggatactt
     1381 cccagccctg ggatgcctgc gctgctctcc ctcgtgagcc ttctctccgt gctgctgatg
     1441 ggttgcgtag ctgactcaga agtcaatcaa gaagctaagc cagaggtcaa gcccgaagtc
     1501 aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagagat cttcttcaag
     1561 atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag
     1621 gaaatggact ccttaacgtt cttgtacgac ggtattgaaa ttcaagctga tcagacccct
     1681 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggaggt
     1741 ggttctctgg aggtgctgtt tcagggcccc gaggaacctc tagtggtgaa ggtggaagag
     1801 ggagataacg ctgtgctgca gtgcctcaag gggacctcag atggccccac tcagcagctg
     1861 acctggtctc gggagtcccc gcttaaaccc ttcttaaaac tcagcctggg gctgccaggc
     1921 ctgggaatcc acgtgagtcc cctggccatc tggcttttca tctccaacgt ctctcaacag
     1981 atggggggct tctacctgtg ccagccgggg cccccctctg agaaggcctg gcagcctggc
     2041 tggacagtca atgtggaggg cagcggggag ctgttccggt ggaatgtttc ggacctaggt
     2101 ggcctgggct gtggcctgaa gaacaggtcc tcagagggcc ccagctcccc ttccgggaag
     2161 ctcatgagcc ccaagctgta tgtgtgggcc aaagaccgcc ctgagatctg ggagggagag
     2221 cctccgtgtc tcccaccgag ggacagcctg aaccagagcc tcagccagga cctcaccatg
     2281 gcccctggct ccacactctg gctgtcctgt ggggtacccc ctgactctgt gtccaggggc
     2341 cccctctcct ggacccatgt gcaccccaag gggcctaagt cattgctgag cctggagctg
     2401 aaggacgatc gcccggccag agatatgtgg gtaatggaga cgggtctgtt gttgccccgg
     2461 gccacagctc aagacgctgg aaagtattat tgtcaccgtg gcaacctgac catgtcattc
     2521 cacctggaga tcactgctcg accaagaggc gtccctcata tcgtgatggt cgacgcctat
     2581 aagaggtata aacatcatca tcatcaccat taaggatccc ccgacctcga cctctggcta
     2641 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     2701 aaggacatat gggagggcaa atcatttggt cgagatccct cggagatctc tagctagagg
     2761 atcgatcccc gccccggacg aactaaacct gactacgaca tctctgcccc ttcttcgcgg
     2821 ggcagtgcat gtaatccctt cagttggttg gtacaacttg ccaactgggc cctgttccac
     2881 atgtgacacg gggggggacc aaacacaaag gggttctctg actgtagttg acatccttat
     2941 aaatggatgt gcacatttgc caacactgag tggctttcat cctggagcag actttgcagt
     3001 ctgtggactg caacacaaca ttgcctttat gtgtaactct tggctgaagc tcttacacca
     3061 atgctggggg acatgtacct cccaggggcc caggaagact acgggaggct acaccaacgt
     3121 caatcagagg ggcctgtgta gctaccgata agcggaccct caagagggca ttagcaatag
     3181 tgtttataag gcccccttgt taaccctaaa cgggtagcat atgcttcccg ggtagtagta
     3241 tatactatcc agactaaccc taattcaata gcatatgtta cccaacggga agcatatgct
     3301 atcgaattag ggttagtaaa agggtcctaa ggaacagcga tatctcccac cccatgagct
     3361 gtcacggttt tatttacatg gggtcaggat tccacgaggg tagtgaacca ttttagtcac
     3421 aagggcagtg gctgaagatc aaggagcggg cagtgaactc tcctgaatct tcgcctgctt
     3481 cttcattctc cttcgtttag ctaatagaat aactgctgag ttgtgaacag taaggtgtat
     3541 gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag aataaaattt ggacgggggg
     3601 ttcagtggtg gcattgtgct atgacaccaa tataaccctc acaaacccct tgggcaataa
     3661 atactagtgt aggaatgaaa cattctgaat atctttaaca atagaaatcc atggggtggg
     3721 gacaagccgt aaagactgga tgtccatctc acacgaattt atggctatgg gcaacacata
     3781 atcctagtgc aatatgatac tggggttatt aagatgtgtc ccaggcaggg accaagacag
     3841 gtgaaccatg ttgttacact ctatttgtaa caaggggaaa gagagtggac gccgacagca
     3901 gcggactcca ctggttgtct ctaacacccc cgaaaattaa acggggctcc acgccaatgg
     3961 ggcccataaa caaagacaag tggccactct tttttttgaa attgtggagt gggggcacgc
     4021 gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa ataagggtgt aataacttgg
     4081 ctgattgtaa ccccgctaac cactgcggtc aaaccacttg cccacaaaac cactaatggc
     4141 accccgggga atacctgcat aagtaggtgg gcgggccaag ataggggcgc gattgctgcg
     4201 atctggagga caaattacac acacttgcgc ctgagcgcca agcacagggt tgttggtcct
     4261 catattcacg aggtcgctga gagcacggtg ggctaatgtt gccatgggta gcatatacta
     4321 cccaaatatc tggatagcat atgctatcct aatctatatc tgggtagcat aggctatcct
     4381 aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagtat atgctatcct
     4441 aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct
     4501 aatctatatc tgggtagtat atgctatcct aatctgtatc cgggtagcat atgctatcct
     4561 catgataagc tgtcaaacat gagaattttc ttgaagacga aagggcctcg tgatacgcct
     4621 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4681 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4741 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4801 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4861 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4921 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4981 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     5041 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5101 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5161 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5221 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5281 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     5341 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5401 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5461 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5521 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5581 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5641 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5701 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5761 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5821 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5881 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5941 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6001 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6061 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6121 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6181 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6241 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6301 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6361 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6421 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6481 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6541 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6601 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     6661 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     6721 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     6781 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct
     6841 agaggtcgac caattctcat gtttgacagc tta
//
