LOCUS       sequence-304353-        7287 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 7287)
  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 7287)
  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-PVR(FL)-FLAG
FEATURES             Location/Qualifiers
     source          1..7287
                     /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             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1302..1325
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1382..1437
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1436..1455)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3673..3691)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3759..3863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3864..3932,3933..4724)
                     /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(4082..4101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4895..5483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5384..5403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5637..5654
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5771..5792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5807..5812,5813..5830,5831..5837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5845..5861
                     /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     5850..5872
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5869..5885
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5869..5885
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6156..6535
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6536..6739
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6690..6710
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6736..6760
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     7241..7260
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gcggccgcgc tagcgccacc atggctagag ctatggctgc tgcttggcct ctgctgctgg
       61 ttgctctgct ggttctttct tggcctccac ctggctctca ccaccaccat caccatggaa
      121 ctggggatgt tgtggtgcag gctcctacac aggtgccagg ctttctgggc gattctgtga
      181 ccctgccttg ctacctgcaa gtgcccaaca tggaagtgac ccacgtgtcc cagctgacat
      241 gggccagaca tggcgaatct ggcagcatgg ccgtgtttca ccagacacag ggccctagct
      301 acagcgagag caagagactg gaattcgtgg ccgccagact gggcgctgaa ctgagaaatg
      361 ccagcctgag aatgttcggc ctgagagtgg aagatgaggg caactacacc tgtctgttcg
      421 tgacctttcc acagggcagc agaagcgtgg acatctggct gagagtgctg gccaaacctc
      481 agaataccgc cgaggtgcag aaagtgcagc tgacaggcga acctgtgcct atggccagat
      541 gtgtgtctac tggtggcaga cctcctgctc agatcacctg gcattctgat ctcggcggca
      601 tgcccaatac ttctcaggtg cccggattcc tgagcggcac agtgacagtg acaagcctgt
      661 ggatcctggt gcctagctct caggtggacg gaaagaacgt gacctgcaag gtggaacacg
      721 agagcttcga gaagcctcag ctgctgaccg tgaacctgac cgtgtactat cctcctgagg
      781 tgtccatcag cggctacgac aacaactggt atctgggcca gaacgaggcc acactgacct
      841 gcgacgccag aagcaatcct gagcctaccg gctacaactg gtccaccaca atgggccctc
      901 tgcctccatt tgctgttgct caaggcgccc agctgctcat cagacccgtg gacaagccca
      961 tcaacacaac cctgatctgc aacgtgacaa acgccctggg agccagacag gctgagctga
     1021 cagtgcaagt gaaagaggga cctcctagcg agcacagcgg catcagcaga aacgccatca
     1081 tctttctggt gctgggcatc ctggtgtttc tgatcctgct cggcatcggc atctacttct
     1141 actggtccaa gtgcagccgg gaagtgctgt ggcattgtca cctgtgtcct agctctacag
     1201 agcatgccag cgcctctgcc aatggccatg tgtcttatag cgccgtgtcc agagagaaca
     1261 gcagctccca ggatcctcag acagagggca caagaggcag cgactacaag gacgacgacg
     1321 acaagtaaac gcgttctaga cacgtgatta attaaggatc ccccgacctc gacctctggc
     1381 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     1441 ggaaggacat atgggagggc aaatcatttg gtcgagatcc ctcggagatc tctagctaga
     1501 ggatcgatcc ccgccccgga cgaactaaac ctgactacga catctctgcc ccttcttcgc
     1561 ggggcagtgc atgtaatccc ttcagttggt tggtacaact tgccaactgg gccctgttcc
     1621 acatgtgaca cgggggggga ccaaacacaa aggggttctc tgactgtagt tgacatcctt
     1681 ataaatggat gtgcacattt gccaacactg agtggctttc atcctggagc agactttgca
     1741 gtctgtggac tgcaacacaa cattgccttt atgtgtaact cttggctgaa gctcttacac
     1801 caatgctggg ggacatgtac ctcccagggg cccaggaaga ctacgggagg ctacaccaac
     1861 gtcaatcaga ggggcctgtg tagctaccga taagcggacc ctcaagaggg cattagcaat
     1921 agtgtttata aggccccctt gttaacccta aacgggtagc atatgcttcc cgggtagtag
     1981 tatatactat ccagactaac cctaattcaa tagcatatgt tacccaacgg gaagcatatg
     2041 ctatcgaatt agggttagta aaagggtcct aaggaacagc gatatctccc accccatgag
     2101 ctgtcacggt tttatttaca tggggtcagg attccacgag ggtagtgaac cattttagtc
     2161 acaagggcag tggctgaaga tcaaggagcg ggcagtgaac tctcctgaat cttcgcctgc
     2221 ttcttcattc tccttcgttt agctaataga ataactgctg agttgtgaac agtaaggtgt
     2281 atgtgaggtg ctcgaaaaca aggtttcagg tgacgccccc agaataaaat ttggacgggg
     2341 ggttcagtgg tggcattgtg ctatgacacc aatataaccc tcacaaaccc cttgggcaat
     2401 aaatactagt gtaggaatga aacattctga atatctttaa caatagaaat ccatggggtg
     2461 gggacaagcc gtaaagactg gatgtccatc tcacacgaat ttatggctat gggcaacaca
     2521 taatcctagt gcaatatgat actggggtta ttaagatgtg tcccaggcag ggaccaagac
     2581 aggtgaacca tgttgttaca ctctatttgt aacaagggga aagagagtgg acgccgacag
     2641 cagcggactc cactggttgt ctctaacacc cccgaaaatt aaacggggct ccacgccaat
     2701 ggggcccata aacaaagaca agtggccact cttttttttg aaattgtgga gtgggggcac
     2761 gcgtcagccc ccacacgccg ccctgcggtt ttggactgta aaataagggt gtaataactt
     2821 ggctgattgt aaccccgcta accactgcgg tcaaaccact tgcccacaaa accactaatg
     2881 gcaccccggg gaatacctgc ataagtaggt gggcgggcca agataggggc gcgattgctg
     2941 cgatctggag gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc
     3001 ctcatattca cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac
     3061 tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc
     3121 ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc
     3181 ctaatttata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc
     3241 ctaatctata tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc
     3301 ctaatagaga ttagggtagt atatgctatc ctaatttata tctgggtagc atatactacc
     3361 caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatat gctatcctaa
     3421 tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa
     3481 tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa
     3541 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa
     3601 tctgtatccg ggtagcatat gctatcctca tgataagctg tcaaacatga gaattttctt
     3661 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3721 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3781 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3841 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3901 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3961 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4021 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4081 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     4141 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4201 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4261 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4321 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4381 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     4441 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4501 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4561 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4621 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4681 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4741 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4801 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4861 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4921 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4981 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5041 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5101 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5161 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5221 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5281 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5341 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5401 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5461 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5521 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5581 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5641 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5701 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5761 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5821 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5881 atgaccatga ttacgccaag ctctagctag aggtcgacca attctcatgt ttgacagctt
     5941 atcatcgcag atccgggcaa cgttgttgcc attgctgcag gcgcagaact ggtaggtatg
     6001 gaagatctat acattgaatc aatattggca attagccata ttagtcattg gttatatagc
     6061 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
     6121 ttatattggc tcatgtccaa tatgaccgcc atgttgacat tgattattga ctagttatta
     6181 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     6241 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     6301 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     6361 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc
     6421 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     6481 acgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     6541 gcggttttgg cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag
     6601 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     6661 aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga
     6721 ggtctatata agcagagctc gtttagtgaa ccgtcagatc ctcactctct tccgcatcgc
     6781 tgtctgcgag ggccagctgt tgggctcgcg gttgaggaca aactcttcgc ggtctttcca
     6841 gtactcttgg atcggaaacc cgtcggcctc cgaacggtac tccgccaccg agggacctga
     6901 gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt
     6961 cgcaaggtag gctgagcacc gtggcgggcg gcagcgggtg gcggtcgggg ttgtttctgg
     7021 cggaggtgct gctgatgatg taattaaagt aggcggtctt gagacggcgg atggtcgagg
     7081 tgaggtgtgg caggcttgag atccagctgt tggggtgagt actccctctc aaaagcgggc
     7141 attacttctg cgctaagatt gtcagtttcc aaaaacgagg aggatttgat attcacctgg
     7201 cccgatctgg ccatacactt gagtgacaat gacatccact ttgcctttct ctccacaggt
     7261 gtccactccc aggtccaagt ttaaact
//
