LOCUS       sequence-304359-        7080 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 7080)
  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 7080)
  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-KIR2DL1(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             90..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1095..1118
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1175..1230
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1229..1248)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3466..3484)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3552..3656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3657..3725,3726..4517)
                     /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(3875..3894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4688..5276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5177..5196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5430..5447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5564..5585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5600..5605,5606..5623,5624..5630)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5638..5654
                     /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     5643..5665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5662..5678
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5662..5678
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        5949..6328
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6329..6532
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6483..6503
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6529..6553
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     7034..7053
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atgtctctgc tggttgtttc tatggcctgc gtgggattct
       61 tcctgctgca aggcgcttgg cctggatctc accaccatca ccatcatcac gagggcgtgc
      121 acagaaagcc tagcctgctt gctcatcccg gacctctggt caagagcgaa gagacagtga
      181 tcctgcagtg ttggagcgac gtgatgttcg agcactttct gctgcaccgc gagggcatgt
      241 tcaacgacac actgagactg atcggcgagc accacgatgg cgtgtccaag gccaatttca
      301 gcatcagcag aatgacccag gacctggccg gcacctacag atgttatggc tctgtgacac
      361 acagccccta ccaggtgtca gcccctagcg atcctctgga catcgtgatc atcggcctgt
      421 acgagaagcc aagcctgtct gcacagcctg gacctacagt gctggccggc gaaaatgtga
      481 ccctgagctg tagcagccgg tccagctacg atatgtacca cctgagcaga gagggcgaag
      541 cccacgaaag aaggctgcct gccggaccta aagtgaacgg caccttccag gccgattttc
      601 cactgggacc tgccacacac ggcggcacat atagatgctt cggcagcttc cacgacagcc
      661 cttacgagtg gtctaagagc agcgatccac tgctggtgtc cgtgaccggc aatcctagca
      721 atagctggcc ttctccaacc gagcctagca gcaagacagg caaccccaga catctgcaca
      781 tcctgatcgg caccagcgtg gtcatcatcc tgttcatcct gctgttcttt ctgctccaca
      841 gatggtgcag caacaagaaa aacgccgccg tgatggatca agagagcgcc ggcaatagaa
      901 ccgccaacag cgaggattcc gacgagcagg accctcaaga agtgacctac acacagctga
      961 accactgcgt gttcacccag cggaagatca ccagacctag ccagaggcct aagacacctc
     1021 ctaccgacat catcgtgtac accgagctgc ctaacgccga gagcagatcc aaggtggtgt
     1081 cttgccctgg cagcgactac aaggacgacg acgataagta aacgcgttct agacacgtga
     1141 ttaattaagg atcccccgac ctcgacctct ggctaataaa ggaaatttat tttcattgca
     1201 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     1261 ttggtcgaga tccctcggag atctctagct agaggatcga tccccgcccc ggacgaacta
     1321 aacctgacta cgacatctct gccccttctt cgcggggcag tgcatgtaat cccttcagtt
     1381 ggttggtaca acttgccaac tgggccctgt tccacatgtg acacgggggg ggaccaaaca
     1441 caaaggggtt ctctgactgt agttgacatc cttataaatg gatgtgcaca tttgccaaca
     1501 ctgagtggct ttcatcctgg agcagacttt gcagtctgtg gactgcaaca caacattgcc
     1561 tttatgtgta actcttggct gaagctctta caccaatgct gggggacatg tacctcccag
     1621 gggcccagga agactacggg aggctacacc aacgtcaatc agaggggcct gtgtagctac
     1681 cgataagcgg accctcaaga gggcattagc aatagtgttt ataaggcccc cttgttaacc
     1741 ctaaacgggt agcatatgct tcccgggtag tagtatatac tatccagact aaccctaatt
     1801 caatagcata tgttacccaa cgggaagcat atgctatcga attagggtta gtaaaagggt
     1861 cctaaggaac agcgatatct cccaccccat gagctgtcac ggttttattt acatggggtc
     1921 aggattccac gagggtagtg aaccatttta gtcacaaggg cagtggctga agatcaagga
     1981 gcgggcagtg aactctcctg aatcttcgcc tgcttcttca ttctccttcg tttagctaat
     2041 agaataactg ctgagttgtg aacagtaagg tgtatgtgag gtgctcgaaa acaaggtttc
     2101 aggtgacgcc cccagaataa aatttggacg gggggttcag tggtggcatt gtgctatgac
     2161 accaatataa ccctcacaaa ccccttgggc aataaatact agtgtaggaa tgaaacattc
     2221 tgaatatctt taacaataga aatccatggg gtggggacaa gccgtaaaga ctggatgtcc
     2281 atctcacacg aatttatggc tatgggcaac acataatcct agtgcaatat gatactgggg
     2341 ttattaagat gtgtcccagg cagggaccaa gacaggtgaa ccatgttgtt acactctatt
     2401 tgtaacaagg ggaaagagag tggacgccga cagcagcgga ctccactggt tgtctctaac
     2461 acccccgaaa attaaacggg gctccacgcc aatggggccc ataaacaaag acaagtggcc
     2521 actctttttt ttgaaattgt ggagtggggg cacgcgtcag cccccacacg ccgccctgcg
     2581 gttttggact gtaaaataag ggtgtaataa cttggctgat tgtaaccccg ctaaccactg
     2641 cggtcaaacc acttgcccac aaaaccacta atggcacccc ggggaatacc tgcataagta
     2701 ggtgggcggg ccaagatagg ggcgcgattg ctgcgatctg gaggacaaat tacacacact
     2761 tgcgcctgag cgccaagcac agggttgttg gtcctcatat tcacgaggtc gctgagagca
     2821 cggtgggcta atgttgccat gggtagcata tactacccaa atatctggat agcatatgct
     2881 atcctaatct atatctgggt agcataggct atcctaatct atatctgggt agcatatgct
     2941 atcctaatct atatctgggt agtatatgct atcctaattt atatctgggt agcataggct
     3001 atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct
     3061 atcctaatct gtatccgggt agcatatgct atcctaatag agattagggt agtatatgct
     3121 atcctaattt atatctgggt agcatatact acccaaatat ctggatagca tatgctatcc
     3181 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagca taggctatcc
     3241 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc
     3301 taatttatat ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc
     3361 taatctatat ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc
     3421 tcatgataag ctgtcaaaca tgagaatttt cttgaagacg aaagggcctc gtgatacgcc
     3481 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     3541 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3601 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3661 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3721 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3781 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3841 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     3901 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3961 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4021 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4081 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4141 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4201 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4261 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4321 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4381 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4441 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4501 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4561 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4621 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4681 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4741 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4801 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4861 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4921 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4981 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5041 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5101 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5161 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5221 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5281 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5341 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5401 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5461 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5521 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5581 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5641 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc
     5701 tagaggtcga ccaattctca tgtttgacag cttatcatcg cagatccggg caacgttgtt
     5761 gccattgctg caggcgcaga actggtaggt atggaagatc tatacattga atcaatattg
     5821 gcaattagcc atattagtca ttggttatat agcataaatc aatattggct attggccatt
     5881 gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc caatatgacc
     5941 gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     6001 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     6061 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     6121 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     6181 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg
     6241 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat
     6301 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg
     6361 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     6421 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt
     6481 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
     6541 gaaccgtcag atcctcactc tcttccgcat cgctgtctgc gagggccagc tgttgggctc
     6601 gcggttgagg acaaactctt cgcggtcttt ccagtactct tggatcggaa acccgtcggc
     6661 ctccgaacgg tactccgcca ccgagggacc tgagcgagtc cgcatcgacc ggatcggaaa
     6721 acctctcgag aaaggcgtct aaccagtcac agtcgcaagg taggctgagc accgtggcgg
     6781 gcggcagcgg gtggcggtcg gggttgtttc tggcggaggt gctgctgatg atgtaattaa
     6841 agtaggcggt cttgagacgg cggatggtcg aggtgaggtg tggcaggctt gagatccagc
     6901 tgttggggtg agtactccct ctcaaaagcg ggcattactt ctgcgctaag attgtcagtt
     6961 tccaaaaacg aggaggattt gatattcacc tggcccgatc tggccataca cttgagtgac
     7021 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca agtttaaact
//
