LOCUS       sequence-303991-        8602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8602)
  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 8602)
  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: pD649-HAsp-LRRC4C-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     149..168
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      254..842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     743..762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     996..1013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1111..1128
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1112..1128
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1584..1600
                     /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)."
     enhancer        1767..2146
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2147..2350
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2301..2321
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2347..2371
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2581..2599
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2581..2598
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2612..2631
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2835..2852
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             4207..4242
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4252..4275
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5935..5979
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5980..6006
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6146..6163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6152..6263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6381..6401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6386..6715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6566..6701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6628..6647
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6763..6810
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6829..7428
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6829..6848)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7325..7345
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7558..7679
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7595..7614)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7649..7668
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7732..8523,8524..8592))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8355..8374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8593..8602,1..82))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac
      121 attatcgcga gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa
      181 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
      241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      361 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      421 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     1021 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     1081 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg
     1141 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct
     1201 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg
     1261 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa
     1321 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc
     1381 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga
     1441 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc
     1501 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag
     1561 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt
     1621 ggcgttgact caatattggc cattagccat attattcatt ggttatatag cataaatcaa
     1681 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg
     1741 ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc
     1801 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1861 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1921 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1981 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2041 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2101 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2161 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2221 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2281 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2341 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     2401 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac
     2461 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc
     2521 cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     2581 atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc
     2641 caggtccaac tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc
     2701 atcattgccc tgagctacat cttctgcctg gtgttcgctg cggccgccca aacatgtcct
     2761 agcgtgtgca gctgtagcaa ccagttcagc aaagtgatct gcgtgcggaa gaacctgaga
     2821 gaggtgccag acggcatcag caccaacacc agactgctga acctgcacga gaaccagatc
     2881 cagatcatca aagtgaacag cttcaagcac ctgaggcacc tggaaatcct gcagctgagc
     2941 cggaaccaca tcagaacaat cgagatcggc gccttcaacg gcctggccaa cctgaatacc
     3001 ctggaactgt tcgacaaccg gctgaccacc attcctaatg gcgccttcgt gtacctgagc
     3061 aagctgaaag agctgtggct gcggaacaac cccatcgagt ctatccccag ctacgccttc
     3121 aatagaatcc ccagcctgag aaggctggac ctgggagagc tgaagagact gagctatatc
     3181 agcgagggcg cctttgaggg cctgagcaac ctcagatacc tgaacctggc catgtgcaat
     3241 ctgagagaga tccccaatct gacccctctg atcaagctgg acgagctgga tctgagcggc
     3301 aaccacctgt ctgccatcag acctggaagt ttccagggcc tgatgcatct gcagaaactg
     3361 tggatgatcc agagccagat ccaagtgatc gagcggaacg ccttcgacaa tctgcagagc
     3421 ctggtggaaa tcaatctggc ccacaacaac ctgacactgc tgccccacga tctgttcaca
     3481 cctctgcacc acctggaacg catccatctg catcacaacc cctggaattg caactgcgac
     3541 atcctgtggc tgagctggtg gatcaaggac atggccccta gcaataccgc ctgctgcgcc
     3601 agatgcaaca cccctcctaa cctgaagggc agatatatcg gcgagctgga ccagaactac
     3661 ttcacctgtt acgcccctgt gatcgtggaa cctcctgccg acctgaatgt gacagaaggc
     3721 atggccgccg agctgaagtg tagagcctct accagcctga ccagcgtgtc ctggatcacc
     3781 cctaatggca cagtgatgac ccacggcgcc tacaaagtgc ggattgccgt gctgtccgac
     3841 ggcaccctga atttcacaaa cgtgaccgtg caggacaccg gcatgtacac ctgtatggtg
     3901 tccaacagcg tgggcaatac cacagccagc gccacactga acgtgacagc cgccacaacc
     3961 acacctttca gctacttcag caccgtgaca gtggaaacca tggaacccag ccaggatgag
     4021 gccagaacca ccgataacaa cgtgggccct acacctgtgg tggactggga aaccaccaac
     4081 gtgaccacaa gcctgacacc tcagagcacc agaagcaccg agaaaacctt cacaatcccc
     4141 gtgaccgaca tcaacagcgg catccctggc atcgacgaag tgatgaagac caccaaaggc
     4201 gcgccggaag atcaagttga tccaagattg attgatggca agggcggatc tctggaggtg
     4261 ctgttccagg gaccaggcgg aagcggcaat cccggcggcg atctggcccc ccagatgctg
     4321 cgcgagctgc aggagaccaa tgccgccctg caggacgtgc gcgagctgct gcgccaccgc
     4381 gtgaaggaga tcaccttcct gaagaacacc gtgatggagt gcgacgcctg cggtatgcag
     4441 ccctctagag gctccggctc catcatccca gttgaggagg agaacccgga cttctggaac
     4501 cgcgaggcag ccgaggccct gggtgccgcc aagaagctgc agcctgcaca gacagccgcc
     4561 aagaacctca tcatcttcct gggcgatggg atgggggtgt ctacggtgac agctgccagg
     4621 atcctaaaag ggcagaagaa ggacaaactg gggcctgaga tacccctggc catggaccgc
     4681 ttcccatatg tggctctgtc caagacatac aatgtagaca aacatgtgcc agacagtgga
     4741 gccacagcca cggcctacct gtgcggggtc aagggcaact tccagaccat tggcttgagt
     4801 gcagccgccc gctttaacca gtgcaacacg acacgcggca acgaggtcat ctccgtgatg
     4861 aatcgggcca agaaagcagg gaagtcagtg ggagtggtaa ccaccacacg agtgcagcac
     4921 gcctcgccag ccggcaccta cgcccacacg gtgaaccgca actggtactc ggacgccgac
     4981 gtgcctgcct cggcccgcca ggaggggtgc caggacatcg ctacgcagct catctccaac
     5041 atggacattg acgtgatcct aggtggaggc cgaaagtaca tgtttcgcat gggaacccca
     5101 gaccctgagt acccagatga ctacagccaa ggtgggacca ggctggacgg gaagaatctg
     5161 gtgcaggaat ggctggcgaa gcgccagggt gcccggtatg tgtggaaccg cactgagctc
     5221 atgcaggctt ccctggaccc gtctgtgacc catctcatgg gtctctttga gcctggagac
     5281 atgaaatacg agatccaccg agactccaca ctggacccct ccctgatgga gatgacagag
     5341 gctgccctgc gcctgctgag caggaacccc cgcggcttct tcctcttcgt ggagggtggt
     5401 cgcatcgacc atggtcatca tgaaagcagg gcttaccggg cactgactga gacgatcatg
     5461 ttcgacgacg ccattgagag ggcgggccag ctcaccagcg aggaggacac gctgagcctc
     5521 gtcactgccg accactccca cgtcttctcc ttcggaggct accccctgcg agggagctcc
     5581 atcttcgggc tggcccctgg caaggcccgg gacaggaagg cctacacggt cctcctatac
     5641 ggaaacggtc caggctatgt gctcaaggac ggcgcccggc cggatgttac cgagagcgag
     5701 agcgggagcc ccgagtatcg gcagcagtca gcagtgcccc tggacgaaga gacccacgca
     5761 ggcgaggacg tggcggtgtt cgcgcgcggc ccgcaggcgc acctggttca cggcgtgcag
     5821 gagcagacct tcatagcgca cgtcatggcc ttcgccgcct gcctggagcc ctacaccgcc
     5881 tgcgacctgg cgccccccgc cggcaccacc gacgccgcgc acccgggtgg cggaggactg
     5941 aacgacatct tcgaggctca gaaaatcgaa tggcacgaac atcaccatca ccatcaccat
     6001 caccattaag gccggcctgt agtcactggc acaacgcgtt acccggtaag ccaatcgggt
     6061 atacacggtc gtcatactgc agacagggtt cttctacttt gcaagatagt cttgagtagt
     6121 aaaataaata gatagagaaa atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6181 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6241 taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat tcttttgatt
     6301 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6361 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     6421 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     6481 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6541 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6601 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     6661 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6721 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
     6781 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gaccgagtac
     6841 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     6901 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     6961 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     7021 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7081 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7141 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7201 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7261 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     7321 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7381 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga
     7441 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     7501 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     7561 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     7621 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7681 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc attaccaatg
     7741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     7861 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     7921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     8041 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8581 ttgaatactc atattcttcc tt
//
