LOCUS       sequence-304357-        7167 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 7167)
  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 7167)
  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-KIR2DL4(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..7167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             96..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1182..1205
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1262..1317
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1316..1335)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3553..3571)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3639..3743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3744..3812,3813..4604)
                     /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(3962..3981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4775..5363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5264..5283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5517..5534
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5651..5672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5687..5692,5693..5710,5711..5717)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5725..5741
                     /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     5730..5752
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5749..5765
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5749..5765
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6036..6415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6416..6619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6570..6590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6616..6640
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     7121..7140
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atgtctatga gccctacagt gatcatcctg gcctgcctgg
       61 gattcttcct ggatcagtct gtgtgggccg gctctcacca ccatcaccat catcacgttg
      121 gcggccagga caagcctttt tgttctgcct ggcctagcgc tgtggtgcct caaggtggac
      181 acgtgaccct gagatgccac tacagacggg gcttcaacat cttcaccctg tacaagaaag
      241 acggcgtgcc cgtgcctgag ctgtacaaca gaatcttctg gaacagcttc ctgatcagcc
      301 ccgtgacacc agctcacgcc ggcacataca gatgcagagg ctttcaccct cacagcccca
      361 cagagtggtc cgctccatct aaccctctgg tcatcatggt caccggcctg tacgagaagc
      421 ctagcctgac agctagaccc ggacctacag ttagagccgg cgagaatgtg acactgagct
      481 gcagcagcca gagcagcttc gacatctacc acctgagcag agagggcgaa gcccacgaac
      541 ttagactgcc tgccgtgcct agcatcaacg gcacctttca ggccgatttt ccactgggac
      601 ctgccacaca cggcgagact tacagatgct ttggcagctt ccacggcagc ccttacgagt
      661 ggtctgatcc tagcgatcct ctgcctgtgt ccgtgacagg caatcctagc agcagctggc
      721 cttctccaac cgagcctagc tttaagaccg gaatcgccag acatctgcac gccgtgatca
      781 gatacagcgt ggccatcatc ctgtttacga tcctgccttt cttcctgctg caccggtggt
      841 gctccaagaa aaaggacgcc gccgtgatga atcaagagcc tgccggccac agaaccgtga
      901 acagagaaga tagcgacgag caggaccctc aagaagtgac atacgcccag ctggaccact
      961 gcatctttac ccagagaaag atcacaggcc ccagccagag aagcaagagg ccttctaccg
     1021 ataccagcgt gtgcatcgag ctgcccaatg ccgaacctag agcactgagc cctgctcacg
     1081 aacaccattc tcaggccctg atgggcagca gcagagagac aacagctctg tctcagaccc
     1141 agctggcttc ctctaatgtg cctgccgccg gaatcggcag cgactacaag gacgacgacg
     1201 acaagtaaac gcgttctaga cacgtgatta attaaggatc ccccgacctc gacctctggc
     1261 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     1321 ggaaggacat atgggagggc aaatcatttg gtcgagatcc ctcggagatc tctagctaga
     1381 ggatcgatcc ccgccccgga cgaactaaac ctgactacga catctctgcc ccttcttcgc
     1441 ggggcagtgc atgtaatccc ttcagttggt tggtacaact tgccaactgg gccctgttcc
     1501 acatgtgaca cgggggggga ccaaacacaa aggggttctc tgactgtagt tgacatcctt
     1561 ataaatggat gtgcacattt gccaacactg agtggctttc atcctggagc agactttgca
     1621 gtctgtggac tgcaacacaa cattgccttt atgtgtaact cttggctgaa gctcttacac
     1681 caatgctggg ggacatgtac ctcccagggg cccaggaaga ctacgggagg ctacaccaac
     1741 gtcaatcaga ggggcctgtg tagctaccga taagcggacc ctcaagaggg cattagcaat
     1801 agtgtttata aggccccctt gttaacccta aacgggtagc atatgcttcc cgggtagtag
     1861 tatatactat ccagactaac cctaattcaa tagcatatgt tacccaacgg gaagcatatg
     1921 ctatcgaatt agggttagta aaagggtcct aaggaacagc gatatctccc accccatgag
     1981 ctgtcacggt tttatttaca tggggtcagg attccacgag ggtagtgaac cattttagtc
     2041 acaagggcag tggctgaaga tcaaggagcg ggcagtgaac tctcctgaat cttcgcctgc
     2101 ttcttcattc tccttcgttt agctaataga ataactgctg agttgtgaac agtaaggtgt
     2161 atgtgaggtg ctcgaaaaca aggtttcagg tgacgccccc agaataaaat ttggacgggg
     2221 ggttcagtgg tggcattgtg ctatgacacc aatataaccc tcacaaaccc cttgggcaat
     2281 aaatactagt gtaggaatga aacattctga atatctttaa caatagaaat ccatggggtg
     2341 gggacaagcc gtaaagactg gatgtccatc tcacacgaat ttatggctat gggcaacaca
     2401 taatcctagt gcaatatgat actggggtta ttaagatgtg tcccaggcag ggaccaagac
     2461 aggtgaacca tgttgttaca ctctatttgt aacaagggga aagagagtgg acgccgacag
     2521 cagcggactc cactggttgt ctctaacacc cccgaaaatt aaacggggct ccacgccaat
     2581 ggggcccata aacaaagaca agtggccact cttttttttg aaattgtgga gtgggggcac
     2641 gcgtcagccc ccacacgccg ccctgcggtt ttggactgta aaataagggt gtaataactt
     2701 ggctgattgt aaccccgcta accactgcgg tcaaaccact tgcccacaaa accactaatg
     2761 gcaccccggg gaatacctgc ataagtaggt gggcgggcca agataggggc gcgattgctg
     2821 cgatctggag gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc
     2881 ctcatattca cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac
     2941 tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc
     3001 ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc
     3061 ctaatttata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc
     3121 ctaatctata tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc
     3181 ctaatagaga ttagggtagt atatgctatc ctaatttata tctgggtagc atatactacc
     3241 caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatat gctatcctaa
     3301 tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa
     3361 tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa
     3421 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa
     3481 tctgtatccg ggtagcatat gctatcctca tgataagctg tcaaacatga gaattttctt
     3541 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3601 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3661 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3721 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3961 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     4021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4261 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     4321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4681 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4741 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4801 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4861 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4921 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4981 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5041 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5101 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5161 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5221 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5281 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5341 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5401 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5461 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5521 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5581 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5641 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5701 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5761 atgaccatga ttacgccaag ctctagctag aggtcgacca attctcatgt ttgacagctt
     5821 atcatcgcag atccgggcaa cgttgttgcc attgctgcag gcgcagaact ggtaggtatg
     5881 gaagatctat acattgaatc aatattggca attagccata ttagtcattg gttatatagc
     5941 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
     6001 ttatattggc tcatgtccaa tatgaccgcc atgttgacat tgattattga ctagttatta
     6061 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     6121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     6181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     6241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc
     6301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     6361 acgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     6421 gcggttttgg cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag
     6481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     6541 aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga
     6601 ggtctatata agcagagctc gtttagtgaa ccgtcagatc ctcactctct tccgcatcgc
     6661 tgtctgcgag ggccagctgt tgggctcgcg gttgaggaca aactcttcgc ggtctttcca
     6721 gtactcttgg atcggaaacc cgtcggcctc cgaacggtac tccgccaccg agggacctga
     6781 gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt
     6841 cgcaaggtag gctgagcacc gtggcgggcg gcagcgggtg gcggtcgggg ttgtttctgg
     6901 cggaggtgct gctgatgatg taattaaagt aggcggtctt gagacggcgg atggtcgagg
     6961 tgaggtgtgg caggcttgag atccagctgt tggggtgagt actccctctc aaaagcgggc
     7021 attacttctg cgctaagatt gtcagtttcc aaaaacgagg aggatttgat attcacctgg
     7081 cccgatctgg ccatacactt gagtgacaat gacatccact ttgcctttct ctccacaggt
     7141 gtccactccc aggtccaagt ttaaact
//
