LOCUS       sequence-304351-        9450 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 9450)
  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 9450)
  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-TIE1(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..9450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      15..24
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             90..107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3465..3488
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3545..3600
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3599..3618)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5836..5854)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5922..6026
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6027..6095,6096..6887)
                     /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(6245..6264)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7058..7646
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7547..7566
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7800..7817
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7934..7955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7970..7975,7976..7993,7994..8000)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8008..8024
                     /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     8013..8035
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8032..8048
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8032..8048
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        8319..8698
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8699..8902
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8853..8873
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     8899..8923
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     9404..9423
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atggtttgga gagttcctcc attcctgctg cctatcctgt
       61 tcctggcctc tcacgttgga gccggatctc accatcacca ccatcatgcc gtggatctga
      121 ccctgctggc caacctgaga ctgaccgatc ctcagagatt cttcctgacc tgtgtgtctg
      181 gcgaagccgg cgctggaaga ggatctgatg cttggggacc tcctctgctg ctggaaaagg
      241 acgacagaat cgtgcggaca cctcctggac ctccactgag actggccaga aatggcagcc
      301 accaagtgac cctgagaggc ttcagcaagc cttctgatct cgtgggcgtg ttctcttgtg
      361 ttggaggcgc tggcgctcgg agaaccagag tgatctatgt gcacaacagc cctggcgctc
      421 atctgctgcc tgacaaagtg acccacaccg tgaacaaggg cgatacagcc gtgctgtccg
      481 ccagagtgca caaagaaaag cagaccgacg tgatctggaa gtccaacggc agctacttct
      541 acaccctgga ttggcacgag gcccaggacg gaagatttct gctgcagctg cctaacgtgc
      601 agcctcctag ctctggcatc tacagcgcca catacctgga agcaagccct ctgggcagcg
      661 ccttcttcag actgatcgtt agaggatgcg gagccggcag atggggccct ggatgtacaa
      721 aagaatgccc tggctgtctg cacggcggcg tgtgtcacga tcatgatggc gagtgtgtgt
      781 gcccacctgg ctttacaggc accagatgtg aacaggcctg cagagagggc agattcggcc
      841 agtcttgtca agagcagtgt cccggcatct ctggctgcag aggcctgacc ttttgcctgc
      901 ctgatcctta cggctgtagc tgtggatctg gatggcgggg ctctcagtgt caagaggctt
      961 gtgcccctgg acactttggc gctgattgca gactgcagtg ccagtgccag aatggcggca
     1021 cctgtgatag attcagcggc tgtgtgtgtc cttctggctg gcatggcgtg cactgcgaga
     1081 agtccgatag aatccctcag atcctgaaca tggccagcga gctggaattc aacctggaaa
     1141 ccatgcctcg gatcaactgc gccgctgccg gaaatccatt tcctgtgcgg ggatccatcg
     1201 agctgagaaa gcctgatggc accgtgctgc tgagcaccaa ggccatcgtg gaacctgaga
     1261 aaaccaccgc cgagttcgag gtgccaagac tggttctggc cgatagcggc ttctgggagt
     1321 gtagagtgtc tacctctggc ggccaggaca gcagacggtt caaagtgaat gtgaaggtgc
     1381 cacctgtgcc tctggcagcc cctagactgc tgacaaaaca gagcagacag ctggtggtgt
     1441 cccctctggt gtcttttagc ggagatggcc ctatcagcac agtgcggctg cactacagac
     1501 cccaggatag cacaatggac tggtctacca tcgtggtgga ccccagcgag aacgtgacac
     1561 tgatgaacct gaggcctaag accggctaca gcgtcagagt tcaactgagc agacctggcg
     1621 aaggcggaga aggcgcatgg ggaccaccta ctctgatgac cacagattgc cctgagcctc
     1681 tgctccagcc ttggctggaa ggatggcatg tggaaggcac cgacagactg agagtgtctt
     1741 ggagtctgcc actggtgcca ggacctctcg ttggagatgg ctttctgctg agactgtggg
     1801 acggcaccag aggccaagag agaagagaga acgtcagcag ccctcaggcc agaacagctc
     1861 tgctgacagg actgacacct ggcacacact accagctgga tgtgcagctg taccactgta
     1921 cactgctggg acctgcctct cctcctgctc atgttctgtt gcctccatct ggccctccag
     1981 ctcctagaca tctgcatgcc caggctctga gcgatagcga gatccagctg acatggaagc
     2041 acccagaagc tctgcccgga ccaatcagca aatacgtggt ggaagtgcag gttgccggcg
     2101 gagctggcga tcctctgtgg attgatgtgg acagacccga ggaaaccagc accatcatca
     2161 ggggcctgaa cgccagcacc agatacctgt tcagaatgcg ggcctctatc caaggcctcg
     2221 gcgattggtc taacaccgtg gaagagtcta ccctcggcaa cggactgcag gctgaaggac
     2281 ctgtgcaaga aagcagagcc gccgaggaag gactggacca gcaactgatt ctggctgtcg
     2341 tgggctccgt gtctgccaca tgtctgacaa ttctggccgc actgctgacc ctcgtgtgca
     2401 tcagaagaag ctgcctgcac agacggcgga cctttaccta tcagtctggc tccggcgagg
     2461 aaacaatcct gcagtttagc agcggcaccc tgacactgac cagaaggcca aaactgcagc
     2521 ccgagccact gagctatcct gtgctggaat gggaagatat caccttcgag gacctgatcg
     2581 gcgagggcaa ctttggccaa gtgatccggg ccatgatcaa gaaagacggc ctgaagatga
     2641 acgccgccat caagatgctg aaagagtacg cctccgagaa cgaccacaga gactttgccg
     2701 gcgaactgga agtgctgtgc aagctgggcc accatcctaa catcatcaat ctgctgggcg
     2761 cctgcaagaa ccggggctac ctgtatatcg ccatcgagta cgccccttac ggaaacctgc
     2821 tggactttct gcggaagtcc agagtgctgg aaacagaccc tgcctttgcc agagagcacg
     2881 gcacagcttc tacactgagt agccggcagc tgctgagatt tgcctctgat gccgccaacg
     2941 gcatgcagta cctgagcgag aagcagttca ttcaccgcga cctggccgct agaaatgtgc
     3001 tcgtgggaga gaacctggcc agcaagatcg ccgattttgg cctgagtaga ggcgaagagg
     3061 tgtacgtgaa gaaaaccatg ggcagactgc ccgtgcggtg gatggccatt gagagcctga
     3121 attacagcgt gtacaccacc aagagcgacg tgtggtcctt tggagtgctg ctgtgggaga
     3181 tcgtgtctct cggcggcaca ccttactgtg gcatgacatg tgccgagctg tacgagaaac
     3241 tgccccaggg ctacagaatg gaacagccca gaaactgcga cgacgaagtg tacgagctga
     3301 tgcggcagtg ttggagagac agaccttacg agaggcctcc attcgctcag atcgctctgc
     3361 agctgggaag aatgctggaa gccagaaagg cctacgtgaa catgtccctg ttcgagaact
     3421 tcacctacgc cggaatcgac gccacagccg aagaggccgg cagcgactac aaggacgacg
     3481 acgataagta aacgcgttct agacacgtga ttaattaagg atcccccgac ctcgacctct
     3541 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     3601 ctcggaagga catatgggag ggcaaatcat ttggtcgaga tccctcggag atctctagct
     3661 agaggatcga tccccgcccc ggacgaacta aacctgacta cgacatctct gccccttctt
     3721 cgcggggcag tgcatgtaat cccttcagtt ggttggtaca acttgccaac tgggccctgt
     3781 tccacatgtg acacgggggg ggaccaaaca caaaggggtt ctctgactgt agttgacatc
     3841 cttataaatg gatgtgcaca tttgccaaca ctgagtggct ttcatcctgg agcagacttt
     3901 gcagtctgtg gactgcaaca caacattgcc tttatgtgta actcttggct gaagctctta
     3961 caccaatgct gggggacatg tacctcccag gggcccagga agactacggg aggctacacc
     4021 aacgtcaatc agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc
     4081 aatagtgttt ataaggcccc cttgttaacc ctaaacgggt agcatatgct tcccgggtag
     4141 tagtatatac tatccagact aaccctaatt caatagcata tgttacccaa cgggaagcat
     4201 atgctatcga attagggtta gtaaaagggt cctaaggaac agcgatatct cccaccccat
     4261 gagctgtcac ggttttattt acatggggtc aggattccac gagggtagtg aaccatttta
     4321 gtcacaaggg cagtggctga agatcaagga gcgggcagtg aactctcctg aatcttcgcc
     4381 tgcttcttca ttctccttcg tttagctaat agaataactg ctgagttgtg aacagtaagg
     4441 tgtatgtgag gtgctcgaaa acaaggtttc aggtgacgcc cccagaataa aatttggacg
     4501 gggggttcag tggtggcatt gtgctatgac accaatataa ccctcacaaa ccccttgggc
     4561 aataaatact agtgtaggaa tgaaacattc tgaatatctt taacaataga aatccatggg
     4621 gtggggacaa gccgtaaaga ctggatgtcc atctcacacg aatttatggc tatgggcaac
     4681 acataatcct agtgcaatat gatactgggg ttattaagat gtgtcccagg cagggaccaa
     4741 gacaggtgaa ccatgttgtt acactctatt tgtaacaagg ggaaagagag tggacgccga
     4801 cagcagcgga ctccactggt tgtctctaac acccccgaaa attaaacggg gctccacgcc
     4861 aatggggccc ataaacaaag acaagtggcc actctttttt ttgaaattgt ggagtggggg
     4921 cacgcgtcag cccccacacg ccgccctgcg gttttggact gtaaaataag ggtgtaataa
     4981 cttggctgat tgtaaccccg ctaaccactg cggtcaaacc acttgcccac aaaaccacta
     5041 atggcacccc ggggaatacc tgcataagta ggtgggcggg ccaagatagg ggcgcgattg
     5101 ctgcgatctg gaggacaaat tacacacact tgcgcctgag cgccaagcac agggttgttg
     5161 gtcctcatat tcacgaggtc gctgagagca cggtgggcta atgttgccat gggtagcata
     5221 tactacccaa atatctggat agcatatgct atcctaatct atatctgggt agcataggct
     5281 atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct
     5341 atcctaattt atatctgggt agcataggct atcctaatct atatctgggt agcatatgct
     5401 atcctaatct atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct
     5461 atcctaatag agattagggt agtatatgct atcctaattt atatctgggt agcatatact
     5521 acccaaatat ctggatagca tatgctatcc taatctatat ctgggtagca tatgctatcc
     5581 taatctatat ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc
     5641 taatctatat ctgggtagta tatgctatcc taatttatat ctgggtagca taggctatcc
     5701 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc
     5761 taatctgtat ccgggtagca tatgctatcc tcatgataag ctgtcaaaca tgagaatttt
     5821 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5881 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5941 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     6001 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6061 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6121 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6181 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6241 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     6301 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6361 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6421 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6481 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6541 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     6601 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6661 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6721 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6781 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6841 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6901 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6961 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     7021 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7081 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7141 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7201 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7261 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7321 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7381 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7441 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7501 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7561 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7621 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7681 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7741 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7801 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     7861 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     7921 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     7981 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     8041 gctatgacca tgattacgcc aagctctagc tagaggtcga ccaattctca tgtttgacag
     8101 cttatcatcg cagatccggg caacgttgtt gccattgctg caggcgcaga actggtaggt
     8161 atggaagatc tatacattga atcaatattg gcaattagcc atattagtca ttggttatat
     8221 agcataaatc aatattggct attggccatt gcatacgttg tatctatatc ataatatgta
     8281 catttatatt ggctcatgtc caatatgacc gccatgttga cattgattat tgactagtta
     8341 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     8401 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     8461 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     8521 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc
     8581 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     8641 cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     8701 gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc
     8761 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     8821 tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg
     8881 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcctcactc tcttccgcat
     8941 cgctgtctgc gagggccagc tgttgggctc gcggttgagg acaaactctt cgcggtcttt
     9001 ccagtactct tggatcggaa acccgtcggc ctccgaacgg tactccgcca ccgagggacc
     9061 tgagcgagtc cgcatcgacc ggatcggaaa acctctcgag aaaggcgtct aaccagtcac
     9121 agtcgcaagg taggctgagc accgtggcgg gcggcagcgg gtggcggtcg gggttgtttc
     9181 tggcggaggt gctgctgatg atgtaattaa agtaggcggt cttgagacgg cggatggtcg
     9241 aggtgaggtg tggcaggctt gagatccagc tgttggggtg agtactccct ctcaaaagcg
     9301 ggcattactt ctgcgctaag attgtcagtt tccaaaaacg aggaggattt gatattcacc
     9361 tggcccgatc tggccataca cttgagtgac aatgacatcc actttgcctt tctctccaca
     9421 ggtgtccact cccaggtcca agtttaaact
//
