LOCUS       sequence-304349-        8424 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 8424)
  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 8424)
  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-NTRK1(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..8424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             126..143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2439..2462
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    2519..2574
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2573..2592)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4810..4828)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4896..5000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5001..5069,5070..5861)
                     /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(5219..5238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6032..6620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6521..6540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6774..6791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6908..6929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6944..6949,6950..6967,6968..6974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6982..6998
                     /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     6987..7009
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7006..7022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7006..7022
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        7293..7672
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7673..7876
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7827..7847
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7873..7897
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     8378..8397
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atgcttagag gtggaagaag aggacagctc ggctggcatt
       61 cttgggctgc tggacctgga tctctgctgg cttggctgat tcttgcttct gccggcgctg
      121 gaagccacca ccatcaccat catgctccct gtcctgatgc ctgttgtcct cacggaagca
      181 gcggcctgag atgtacaaga gatggcgccc tggacagcct gcatcatctg cctggtgccg
      241 agaatctgac cgagctgtac atcgagaacc agcagcatct gcagcacctg gaactgagag
      301 atctgagagg cctgggcgag ctgagaaacc tgacaatcgt gaagtccggg ctgagattcg
      361 tggcccctga cgcctttcac ttcaccccta gactgagccg gctgaacctg agcttcaatg
      421 ccctggaatc cctgagctgg aaaaccgtgc agggcctgtc tctgcaagag ctggtgctgt
      481 ctggcaatcc tctgcactgt agctgtgccc tgagatggct gcagagatgg gaagaagaag
      541 gcctcggcgg agtccctgag cagaaactgc aatgtcatgg ccagggacct ctggctcaca
      601 tgcctaatgc aagctgtggc gtgcccacac tgaaggtgca ggttccaaat gcctctgtgg
      661 acgtgggaga tgacgtgctg ctgagatgcc aggttgaagg cagaggactg gaacaggccg
      721 gatggatcct gacagagctg gaacagagcg ccaccgtgat gaagtctggt ggactgcctt
      781 ctctgggcct gacactggcc aatgtgacca gcgacctgaa ccggaagaat gtgacctgct
      841 gggccgagaa cgatgtgggc agagctgaag tgtccgtgca agtgaacgtg tcattccctg
      901 ccagcgttca gctgcacaca gccgtggaaa tgcaccactg gtgtatccct ttcagcgtgg
      961 acggacagcc agcacctagc ctgcgttggc tgtttaacgg cagcgtgctg aacgagacaa
     1021 gcttcatctt caccgagttt ctggaaccag ccgccaacga aacagtgcgg catggctgtc
     1081 tgagactgaa ccagcctaca cacgtgaaca acggcaacta caccctgctg gccgccaatc
     1141 cttttggaca ggcctctgcc tctatcatgg ccgccttcat ggacaacccc ttcgagttca
     1201 accccgagga tcccattcct gtgtctttca gccccgtgga caccaatagc acaagcggcg
     1261 accccgtgga aaagaaggat gagacacctt ttggcgtgtc cgtggccgtt ggactggctg
     1321 tgtttgcctg tctgttcctg agcacactgc tgcttgtgct gaacaagtgc ggcagaagaa
     1381 acaagttcgg catcaacaga cctgccgtgc tggctcctga agatggactg gcaatgagcc
     1441 tgcactttat gaccctcggc ggcagcagcc tgtctcctac tgaaggcaaa ggttctggac
     1501 tgcagggcca catcatcgag aatccccagt acttcagcga cgcctgcgtg caccacatca
     1561 agagaagaga catcgtgctg aagtgggaac tcggcgaggg cgcctttgga aaagtgtttc
     1621 tggccgagtg ccacaacctg ctgcctgagc aggataagat gctggtggcc gtgaaggccc
     1681 tgaaagaagc ctctgaaagc gccagacagg acttccagag agaggccgaa ctgctgacaa
     1741 tgctgcaaca ccagcacatc gtgcggttct tcggcgtgtg tacagaaggc agacccctgc
     1801 tgatggtgtt cgagtacatg cggcacggcg atctgaacag attcctgaga agccacggac
     1861 ctgacgccaa acttctggct ggcggagaag atgtggctcc tggacctctt ggactgggac
     1921 agctgctggc agttgcttct caagtggccg ctggaatggt gtatctggcc ggactgcact
     1981 tcgtgcacag agatctggcc accagaaatt gcctcgtcgg acagggcctc gtggtcaaga
     2041 tcggcgattt cggcatgagc cgggacatct acagcaccga ctattacaga gtcggcggac
     2101 ggaccatgct gcccattaga tggatgcctc cagagagcat cctgtaccgg aagttcacca
     2161 ccgagagcga cgtgtggtct ttcggagtgg tgctgtggga gatcttcacc tacggcaagc
     2221 agccctggta tcagctgagc aacacagagg ccatcgactg catcacccag ggaagagaac
     2281 tggaaagacc cagagcctgt cctcctgagg tgtacgccat catgagaggc tgttggcaga
     2341 gagagcctca gcagagacac agcatcaagg acgtgcacgc tagactgcag gcactggctc
     2401 aggctcctcc agtgtacctg gatgtgctcg gcggctccga ctacaaggac gacgatgata
     2461 agtaaacgcg ttctagacac gtgattaatt aaggatcccc cgacctcgac ctctggctaa
     2521 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     2581 aggacatatg ggagggcaaa tcatttggtc gagatccctc ggagatctct agctagagga
     2641 tcgatccccg ccccggacga actaaacctg actacgacat ctctgcccct tcttcgcggg
     2701 gcagtgcatg taatcccttc agttggttgg tacaacttgc caactgggcc ctgttccaca
     2761 tgtgacacgg ggggggacca aacacaaagg ggttctctga ctgtagttga catccttata
     2821 aatggatgtg cacatttgcc aacactgagt ggctttcatc ctggagcaga ctttgcagtc
     2881 tgtggactgc aacacaacat tgcctttatg tgtaactctt ggctgaagct cttacaccaa
     2941 tgctggggga catgtacctc ccaggggccc aggaagacta cgggaggcta caccaacgtc
     3001 aatcagaggg gcctgtgtag ctaccgataa gcggaccctc aagagggcat tagcaatagt
     3061 gtttataagg cccccttgtt aaccctaaac gggtagcata tgcttcccgg gtagtagtat
     3121 atactatcca gactaaccct aattcaatag catatgttac ccaacgggaa gcatatgcta
     3181 tcgaattagg gttagtaaaa gggtcctaag gaacagcgat atctcccacc ccatgagctg
     3241 tcacggtttt atttacatgg ggtcaggatt ccacgagggt agtgaaccat tttagtcaca
     3301 agggcagtgg ctgaagatca aggagcgggc agtgaactct cctgaatctt cgcctgcttc
     3361 ttcattctcc ttcgtttagc taatagaata actgctgagt tgtgaacagt aaggtgtatg
     3421 tgaggtgctc gaaaacaagg tttcaggtga cgcccccaga ataaaatttg gacggggggt
     3481 tcagtggtgg cattgtgcta tgacaccaat ataaccctca caaacccctt gggcaataaa
     3541 tactagtgta ggaatgaaac attctgaata tctttaacaa tagaaatcca tggggtgggg
     3601 acaagccgta aagactggat gtccatctca cacgaattta tggctatggg caacacataa
     3661 tcctagtgca atatgatact ggggttatta agatgtgtcc caggcaggga ccaagacagg
     3721 tgaaccatgt tgttacactc tatttgtaac aaggggaaag agagtggacg ccgacagcag
     3781 cggactccac tggttgtctc taacaccccc gaaaattaaa cggggctcca cgccaatggg
     3841 gcccataaac aaagacaagt ggccactctt ttttttgaaa ttgtggagtg ggggcacgcg
     3901 tcagccccca cacgccgccc tgcggttttg gactgtaaaa taagggtgta ataacttggc
     3961 tgattgtaac cccgctaacc actgcggtca aaccacttgc ccacaaaacc actaatggca
     4021 ccccggggaa tacctgcata agtaggtggg cgggccaaga taggggcgcg attgctgcga
     4081 tctggaggac aaattacaca cacttgcgcc tgagcgccaa gcacagggtt gttggtcctc
     4141 atattcacga ggtcgctgag agcacggtgg gctaatgttg ccatgggtag catatactac
     4201 ccaaatatct ggatagcata tgctatccta atctatatct gggtagcata ggctatccta
     4261 atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta
     4321 atttatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta
     4381 atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata tgctatccta
     4441 atagagatta gggtagtata tgctatccta atttatatct gggtagcata tactacccaa
     4501 atatctggat agcatatgct atcctaatct atatctgggt agcatatgct atcctaatct
     4561 atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct
     4621 atatctgggt agtatatgct atcctaattt atatctgggt agcataggct atcctaatct
     4681 atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaatct
     4741 gtatccgggt agcatatgct atcctcatga taagctgtca aacatgagaa ttttcttgaa
     4801 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4861 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4921 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4981 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5041 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5101 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5161 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5221 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     5281 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5341 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5401 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5461 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5521 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     5581 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5641 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5701 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5761 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5821 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5881 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5941 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6001 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6061 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6181 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6661 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6721 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6781 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6841 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6901 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6961 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7021 accatgatta cgccaagctc tagctagagg tcgaccaatt ctcatgtttg acagcttatc
     7081 atcgcagatc cgggcaacgt tgttgccatt gctgcaggcg cagaactggt aggtatggaa
     7141 gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt atatagcata
     7201 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
     7261 tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta gttattaata
     7321 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     7381 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     7441 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     7501 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
     7561 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
     7621 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     7681 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     7741 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     7801 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
     7861 ctatataagc agagctcgtt tagtgaaccg tcagatcctc actctcttcc gcatcgctgt
     7921 ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac tcttcgcggt ctttccagta
     7981 ctcttggatc ggaaacccgt cggcctccga acggtactcc gccaccgagg gacctgagcg
     8041 agtccgcatc gaccggatcg gaaaacctct cgagaaaggc gtctaaccag tcacagtcgc
     8101 aaggtaggct gagcaccgtg gcgggcggca gcgggtggcg gtcggggttg tttctggcgg
     8161 aggtgctgct gatgatgtaa ttaaagtagg cggtcttgag acggcggatg gtcgaggtga
     8221 ggtgtggcag gcttgagatc cagctgttgg ggtgagtact ccctctcaaa agcgggcatt
     8281 acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc
     8341 gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc cacaggtgtc
     8401 cactcccagg tccaagttta aact
//
