LOCUS       sequence-304361-        8271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell surface expression of His- and FLAG-tagged 
            full-length protein for binding and signaling assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8271)
  AUTHORS   Wojtowicz WM, Vielmetter J, Fernandes RA, Siepe DH, Eastman CL, 
            Chisholm GB, Cox S, Klock H, Anderson PW, Rue SM, Miller JJ, Glaser 
            SM, Bragstad ML, Vance J, Lam AW, Lesley SA, Zinn K, Garcia KC
  TITLE     A Human IgSF Cell-Surface Interactome Reveals a Complex Network of 
            Protein-Protein Interactions.
  JOURNAL   Cell. 2020 Aug 20;182(4):1027-1043.e17. doi: 
            10.1016/j.cell.2020.07.025.
  PUBMED    32822567
REFERENCE   2  (bases 1 to 8271)
  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.cell.2020.07"; journalName: "Cell"; date: "2020-08-20- 
            20"; volume: "182"; issue: "4"; pages: "1027-1043.e17"
COMMENT     Sequence Label: pTT3-SP-6xHis-ISLR2(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..8271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2286..2309
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    2366..2421
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2420..2439)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4657..4675)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4743..4847
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4848..4916,4917..5708)
                     /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(5066..5085)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5879..6467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6368..6387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6621..6638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6755..6776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6791..6796,6797..6814,6815..6821)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6829..6845
                     /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     6834..6856
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6853..6869
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6853..6869
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        7140..7519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7520..7723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7674..7694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7720..7744
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     8225..8244
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atgtttccac tgagagcact ttggctcgtg tgggcccttc
       61 ttggagttgc cggcggatct caccaccacc atcaccactc ttgtcctgag ccttgcgcct
      121 gcgtggacaa atatgcccac cagttcgccg actgcgccta caaagaactg agagaggtgc
      181 cagagggcct gcctgccaat gtgacaacac tgagcctgag cgccaacaag atcaccgtgc
      241 tgagaagagg cgccttcgcc gatgtgacac aagtgacatc tctgtggctg gcccacaacg
      301 aagtgcgaac agttgaacct ggtgctctgg ccgtgctgtc ccagctgaag aatctggacc
      361 tgagccacaa cttcatcagc agcttccctt ggagcgacct gagaaatctg tctgccctgc
      421 agctgctgaa gatgaaccac aacagactgg gcagcctgcc tagagatgct ctgggagcac
      481 tgcctgacct gcggagcctg agaatcaaca acaaccggct gagaacactg gccccaggca
      541 cctttgatgc cctgtctgct ctgagccatc tgcagctgta tcacaaccct ttccactgcg
      601 gctgtggact tgtgtggctt caagcctggg ccgcctctac aagagtgtct ctgccagagc
      661 ctgactctat cgcctgtgct tctccaccag ctctgcaggg cgttccagtg tatagactgc
      721 ctgctctgcc ttgcgctcct ccttctgtgc atctttctgc cgagcctcca ctggaagcac
      781 ctggaacacc tcttagagcc ggactggcct tcgtgctgca ctgtattgcc gatggacacc
      841 ccacacctag actgcagtgg cagcttcaga ttcctggcgg cacagtggtg ctggaacctc
      901 cagttctgtc tggcgaagat gatggcgtgg gagcagaaga aggcgaaggc gaaggcgacg
      961 gggatctgct gacacagaca caggctcaga cacccacacc agctccagct tggcctgctc
     1021 ctcctgccac accaagattt ctggccctgg ccaatggaag cctgctggtt cctctgctgt
     1081 ctgccaaaga ggccggcgtc tacacctgta gagcccacaa tgagctgggc gccaatagca
     1141 cctctattag agtggccgtg gccgccacag gacctccaaa acatgctcct ggtgctggcg
     1201 gagaacctga tggacaggcc cctacaagcg agagaaagag cactgctaaa ggcagaggca
     1261 acagcgtgct gccttctaag cccgagggca agatcaaagg ccagggactc gccaaagtgt
     1321 ccatcctggg agagacagag actgagcccg aagaggatac ctctgagggc gaagaggccg
     1381 aggatcagat tctggctgat cccgccgaag aacagagatg cggcaatggc gatcccagca
     1441 gatacgtgtc caaccacgcc ttcaatcaga gcgccgagct gaagcctcac gtgttcgaac
     1501 tgggagtgat tgccctggat gtggccgaaa gggaagccag agttcagctg acaccactgg
     1561 ctgctagatg gggacctgga ccaggtggcg caggcggagc acctagacct ggaagaaggc
     1621 ctctgagact gctgtatctg tgtccagctg gtggcggagc tgctgtgcaa tggtcaagag
     1681 tggaagaggg cgtgaacgcc tactggttta gaggcctgag gcctggcacc aactacagcg
     1741 tttgtctggc tcttgctggc gaggcctgtc acgtgcaagt ggtgttcagc accaagaaag
     1801 agctgcccag cctgctcgtg attgtggccg tgtctgtttt tctgctggtc ctggctacag
     1861 tgccactgct gggagctgct tgttgccatc tgctggccaa acatcccggc aagccctaca
     1921 gactgattct gaggccacag gctcccgatc ctatggaaaa gagaatcgcc gccgacttcg
     1981 accccagagc cagctacctg gaaagcgaga agtcttatcc agccggcgga gaagctggcg
     2041 gcgaagaacc agaagatgtg caaggcgagg gcctcgatga agatgctgaa caaggcgatc
     2101 ctagcggcga cctgcagaga gaagaatctc tggccgcctg tagcctggtg gaaagccaga
     2161 gcaaggccaa tcaagaggaa ttcgaggccg gcagcgagta ctctgataga cttccactgg
     2221 gagccgaggc cgtgaatatc gcccaagaga tcaacggcaa ctacagacag accgctggcg
     2281 gcagcgacta caaggacgac gatgataagt aaacgcgttc tagacacgtg attaattaag
     2341 gatcccccga cctcgacctc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     2401 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttggtcgag
     2461 atccctcgga gatctctagc tagaggatcg atccccgccc cggacgaact aaacctgact
     2521 acgacatctc tgccccttct tcgcggggca gtgcatgtaa tcccttcagt tggttggtac
     2581 aacttgccaa ctgggccctg ttccacatgt gacacggggg gggaccaaac acaaaggggt
     2641 tctctgactg tagttgacat ccttataaat ggatgtgcac atttgccaac actgagtggc
     2701 tttcatcctg gagcagactt tgcagtctgt ggactgcaac acaacattgc ctttatgtgt
     2761 aactcttggc tgaagctctt acaccaatgc tgggggacat gtacctccca ggggcccagg
     2821 aagactacgg gaggctacac caacgtcaat cagaggggcc tgtgtagcta ccgataagcg
     2881 gaccctcaag agggcattag caatagtgtt tataaggccc ccttgttaac cctaaacggg
     2941 tagcatatgc ttcccgggta gtagtatata ctatccagac taaccctaat tcaatagcat
     3001 atgttaccca acgggaagca tatgctatcg aattagggtt agtaaaaggg tcctaaggaa
     3061 cagcgatatc tcccacccca tgagctgtca cggttttatt tacatggggt caggattcca
     3121 cgagggtagt gaaccatttt agtcacaagg gcagtggctg aagatcaagg agcgggcagt
     3181 gaactctcct gaatcttcgc ctgcttcttc attctccttc gtttagctaa tagaataact
     3241 gctgagttgt gaacagtaag gtgtatgtga ggtgctcgaa aacaaggttt caggtgacgc
     3301 ccccagaata aaatttggac ggggggttca gtggtggcat tgtgctatga caccaatata
     3361 accctcacaa accccttggg caataaatac tagtgtagga atgaaacatt ctgaatatct
     3421 ttaacaatag aaatccatgg ggtggggaca agccgtaaag actggatgtc catctcacac
     3481 gaatttatgg ctatgggcaa cacataatcc tagtgcaata tgatactggg gttattaaga
     3541 tgtgtcccag gcagggacca agacaggtga accatgttgt tacactctat ttgtaacaag
     3601 gggaaagaga gtggacgccg acagcagcgg actccactgg ttgtctctaa cacccccgaa
     3661 aattaaacgg ggctccacgc caatggggcc cataaacaaa gacaagtggc cactcttttt
     3721 tttgaaattg tggagtgggg gcacgcgtca gcccccacac gccgccctgc ggttttggac
     3781 tgtaaaataa gggtgtaata acttggctga ttgtaacccc gctaaccact gcggtcaaac
     3841 cacttgccca caaaaccact aatggcaccc cggggaatac ctgcataagt aggtgggcgg
     3901 gccaagatag gggcgcgatt gctgcgatct ggaggacaaa ttacacacac ttgcgcctga
     3961 gcgccaagca cagggttgtt ggtcctcata ttcacgaggt cgctgagagc acggtgggct
     4021 aatgttgcca tgggtagcat atactaccca aatatctgga tagcatatgc tatcctaatc
     4081 tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc tatcctaatc
     4141 tatatctggg tagtatatgc tatcctaatt tatatctggg tagcataggc tatcctaatc
     4201 tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc tatcctaatc
     4261 tgtatccggg tagcatatgc tatcctaata gagattaggg tagtatatgc tatcctaatt
     4321 tatatctggg tagcatatac tacccaaata tctggatagc atatgctatc ctaatctata
     4381 tctgggtagc atatgctatc ctaatctata tctgggtagc ataggctatc ctaatctata
     4441 tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc ctaatttata
     4501 tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc ctaatctata
     4561 tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc ctcatgataa
     4621 gctgtcaaac atgagaattt tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     4681 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4741 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4801 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4861 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4921 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4981 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5041 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     5101 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5161 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5221 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5281 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5341 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     5401 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5461 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5521 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5581 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5641 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5701 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5761 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5821 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5881 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5941 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6001 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6061 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6121 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6181 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6241 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6301 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6361 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6421 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6481 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6541 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6601 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     6661 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6721 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6781 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6841 aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg
     6901 accaattctc atgtttgaca gcttatcatc gcagatccgg gcaacgttgt tgccattgct
     6961 gcaggcgcag aactggtagg tatggaagat ctatacattg aatcaatatt ggcaattagc
     7021 catattagtc attggttata tagcataaat caatattggc tattggccat tgcatacgtt
     7081 gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac cgccatgttg
     7141 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     7201 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     7261 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     7321 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     7381 agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     7441 gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt
     7501 agtcatcgct attaccatgg tgatgcggtt ttggcagtac accaatgggc gtggatagcg
     7561 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     7621 gcaccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat
     7681 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     7741 gatcctcact ctcttccgca tcgctgtctg cgagggccag ctgttgggct cgcggttgag
     7801 gacaaactct tcgcggtctt tccagtactc ttggatcgga aacccgtcgg cctccgaacg
     7861 gtactccgcc accgagggac ctgagcgagt ccgcatcgac cggatcggaa aacctctcga
     7921 gaaaggcgtc taaccagtca cagtcgcaag gtaggctgag caccgtggcg ggcggcagcg
     7981 ggtggcggtc ggggttgttt ctggcggagg tgctgctgat gatgtaatta aagtaggcgg
     8041 tcttgagacg gcggatggtc gaggtgaggt gtggcaggct tgagatccag ctgttggggt
     8101 gagtactccc tctcaaaagc gggcattact tctgcgctaa gattgtcagt ttccaaaaac
     8161 gaggaggatt tgatattcac ctggcccgat ctggccatac acttgagtga caatgacatc
     8221 cactttgcct ttctctccac aggtgtccac tcccaggtcc aagtttaaac t
//
