LOCUS       sequence-304355-        7161 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 7161)
  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 7161)
  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-KIR2DL5(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             90..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1176..1199
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1256..1311
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1310..1329)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3547..3565)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3633..3737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3738..3806,3807..4598)
                     /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(3956..3975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4769..5357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5258..5277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5511..5528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5645..5666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5681..5686,5687..5704,5705..5711)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5719..5735
                     /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     5724..5746
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5743..5759
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5743..5759
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6030..6409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6410..6613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6564..6584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6610..6634
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     7115..7134
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atgtctctga tggttatttc tatggcctgc gtgggattct
       61 tcctgctgca aggcgcttgg acaggctctc accaccatca ccatcatcac gaaggcggcc
      121 aggataagcc tctgctgtct gcttggcctt ctgctgtggt tcctagaggc ggacacgtga
      181 ccctgctgtg tagaagcaga ctgggcttca ccatcttcag cctgtacaaa gaggacggcg
      241 tccccgttcc tgagctgtac aacaagatct tctggaagtc catcctgatg ggccccgtga
      301 caccagctca cgccggcaca tatagatgca gaggcagcca tcctagaagc cccattgagt
      361 ggtccgctcc tagcaaccct ctggtcatcg tggtcaccgg cctgtttggc aaaccttctc
      421 tgtctgccca gcctggacct acagtgcgga caggcgaaaa tgtgaccctg agctgcagca
      481 gcagatccag cttcgacatg taccacctga gcagagaggg cagagcccac gaacctagac
      541 tgcctgctgt gccttccgtg aatggcacct tccaggccga ttttccactg ggacctgcca
      601 cacacggcgg cacctacaca tgttttggca gcctgcacga cagcccctac gagtggtctg
      661 atccttctga tccactgctg gtgtccgtga ccggcaatag cagtagcagc agctctagcc
      721 ctaccgagcc tagcagcaag accggcattc ggagacatct gcacatcctg atcggcacct
      781 ctgtggccat catcctgttt attatcctgt tcttctttct gctccactgt tgctgctcca
      841 acaagaaaaa cgccgccgtg atggatcaag agcctgccgg cgatagaacc gtgaacagag
      901 aggactctga cgaccaggat cctcaagaag tgacatacgc ccagctggat cactgcgtgt
      961 tcacccagac caagatcaca agccccagcc agaggcctaa gacacctcct accgacacca
     1021 caatgtacat ggaactgccc aacgccaagc ctagatctct gagccctgct cacaaacacc
     1081 acagccaggc tctgagaggc agctccagag aaacaacagc cctgagccag aacagagtgg
     1141 ccagctctca tgtgcctgcc gccggaatcg gcagcgacta caaggacgac gacgacaagt
     1201 aaacgcgttc tagacacgtg attaattaag gatcccccga cctcgacctc tggctaataa
     1261 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     1321 acatatggga gggcaaatca tttggtcgag atccctcgga gatctctagc tagaggatcg
     1381 atccccgccc cggacgaact aaacctgact acgacatctc tgccccttct tcgcggggca
     1441 gtgcatgtaa tcccttcagt tggttggtac aacttgccaa ctgggccctg ttccacatgt
     1501 gacacggggg gggaccaaac acaaaggggt tctctgactg tagttgacat ccttataaat
     1561 ggatgtgcac atttgccaac actgagtggc tttcatcctg gagcagactt tgcagtctgt
     1621 ggactgcaac acaacattgc ctttatgtgt aactcttggc tgaagctctt acaccaatgc
     1681 tgggggacat gtacctccca ggggcccagg aagactacgg gaggctacac caacgtcaat
     1741 cagaggggcc tgtgtagcta ccgataagcg gaccctcaag agggcattag caatagtgtt
     1801 tataaggccc ccttgttaac cctaaacggg tagcatatgc ttcccgggta gtagtatata
     1861 ctatccagac taaccctaat tcaatagcat atgttaccca acgggaagca tatgctatcg
     1921 aattagggtt agtaaaaggg tcctaaggaa cagcgatatc tcccacccca tgagctgtca
     1981 cggttttatt tacatggggt caggattcca cgagggtagt gaaccatttt agtcacaagg
     2041 gcagtggctg aagatcaagg agcgggcagt gaactctcct gaatcttcgc ctgcttcttc
     2101 attctccttc gtttagctaa tagaataact gctgagttgt gaacagtaag gtgtatgtga
     2161 ggtgctcgaa aacaaggttt caggtgacgc ccccagaata aaatttggac ggggggttca
     2221 gtggtggcat tgtgctatga caccaatata accctcacaa accccttggg caataaatac
     2281 tagtgtagga atgaaacatt ctgaatatct ttaacaatag aaatccatgg ggtggggaca
     2341 agccgtaaag actggatgtc catctcacac gaatttatgg ctatgggcaa cacataatcc
     2401 tagtgcaata tgatactggg gttattaaga tgtgtcccag gcagggacca agacaggtga
     2461 accatgttgt tacactctat ttgtaacaag gggaaagaga gtggacgccg acagcagcgg
     2521 actccactgg ttgtctctaa cacccccgaa aattaaacgg ggctccacgc caatggggcc
     2581 cataaacaaa gacaagtggc cactcttttt tttgaaattg tggagtgggg gcacgcgtca
     2641 gcccccacac gccgccctgc ggttttggac tgtaaaataa gggtgtaata acttggctga
     2701 ttgtaacccc gctaaccact gcggtcaaac cacttgccca caaaaccact aatggcaccc
     2761 cggggaatac ctgcataagt aggtgggcgg gccaagatag gggcgcgatt gctgcgatct
     2821 ggaggacaaa ttacacacac ttgcgcctga gcgccaagca cagggttgtt ggtcctcata
     2881 ttcacgaggt cgctgagagc acggtgggct aatgttgcca tgggtagcat atactaccca
     2941 aatatctgga tagcatatgc tatcctaatc tatatctggg tagcataggc tatcctaatc
     3001 tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc tatcctaatt
     3061 tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc tatcctaatc
     3121 tatatctggg tagtatatgc tatcctaatc tgtatccggg tagcatatgc tatcctaata
     3181 gagattaggg tagtatatgc tatcctaatt tatatctggg tagcatatac tacccaaata
     3241 tctggatagc atatgctatc ctaatctata tctgggtagc atatgctatc ctaatctata
     3301 tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc ctaatctata
     3361 tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc ctaatctata
     3421 tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc ctaatctgta
     3481 tccgggtagc atatgctatc ctcatgataa gctgtcaaac atgagaattt tcttgaagac
     3541 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3601 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3661 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3721 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3781 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3841 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3901 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3961 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     4021 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4081 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4141 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4201 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4261 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4321 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4381 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4441 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4501 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4561 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4621 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4681 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4741 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4801 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4861 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4921 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4981 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5041 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5101 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5161 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5221 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5281 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5341 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5401 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5461 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5521 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5581 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     5641 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     5701 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     5761 atgattacgc caagctctag ctagaggtcg accaattctc atgtttgaca gcttatcatc
     5821 gcagatccgg gcaacgttgt tgccattgct gcaggcgcag aactggtagg tatggaagat
     5881 ctatacattg aatcaatatt ggcaattagc catattagtc attggttata tagcataaat
     5941 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
     6001 tggctcatgt ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta
     6061 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     6301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     6361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     6421 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     6481 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     6541 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     6601 tataagcaga gctcgtttag tgaaccgtca gatcctcact ctcttccgca tcgctgtctg
     6661 cgagggccag ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc
     6721 ttggatcgga aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt
     6781 ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc taaccagtca cagtcgcaag
     6841 gtaggctgag caccgtggcg ggcggcagcg ggtggcggtc ggggttgttt ctggcggagg
     6901 tgctgctgat gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gaggtgaggt
     6961 gtggcaggct tgagatccag ctgttggggt gagtactccc tctcaaaagc gggcattact
     7021 tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac ctggcccgat
     7081 ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac aggtgtccac
     7141 tcccaggtcc aagtttaaac t
//
