LOCUS       sequence-303995-        8620 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 8620)
  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 8620)
  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-LRRC4-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8620
                     /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             4225..4260
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4270..4293
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5953..5997
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5998..6024
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6164..6181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6170..6281
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6399..6419)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6404..6733
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6584..6719
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6646..6665
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6781..6828
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6847..7446
                     /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(6847..6866)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7343..7363
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7576..7697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7613..7632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7667..7686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7750..8541,8542..8610))
                     /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     8373..8392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8611..8620,1..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 cggccgccgc atctgctgga
     2761 cctcagaatt gtcctagcgt gtgcagctgc tccaaccagt tcagcaaggt cgtgtgtacc
     2821 agacggggcc tgtctgaagt gcctcagggc atccctagca acaccagata cctgaacctg
     2881 atggaaaaca acatccagat gatccaggcc gacaccttcc ggcatctgca ccatctggaa
     2941 gtgctgcagc tgggcagaaa cagcatccgg cagattgaag tgggcgcctt caatggcctg
     3001 gccagcctga ataccctgga actgttcgac aactggctga cagtgatccc cagcggcgcc
     3061 tttgagtacc tgagcaagct gagagagctg tggctgcgga acaaccccat cgagagcatt
     3121 cccagctacg ccttcaacag agtgcccagc ctgatgagac tggacctggg cgagctgaag
     3181 aagctcgagt acattagcga gggcgctttc gagggcctgt tcaacctgaa gtacctgaat
     3241 ctgggcatgt gcaacatcaa ggacatgccc aatctgaccc ctctcgtggg cctcgaggaa
     3301 ctggaaatga gcggcaatca cttccccgag atcagacccg gctctttcca cggactgagc
     3361 agcctgaaga aactgtgggt catgaacagc caggtgtccc tgatcgagcg gaacgccttt
     3421 gatggactgg cctctctggt ggaactgaac ctggctcaca acaacctgtc cagcctgcct
     3481 cacgacctgt tcacccctct gagatatctg gtcgagctgc atctgcatca caacccctgg
     3541 aattgcgact gcgacatcct gtggctggcc tggtggctca gagagtacat ccccaccaac
     3601 agcacctgtt gcggcagatg tcacgcccct atgcacatga gaggccgcta cctggtggaa
     3661 gtggatcagg ccagcttcca gtgcagcgcc cctttcatta tggacgcccc tcgggacctg
     3721 aacatctccg agggaagaat ggccgagctg aaatgcagaa cccctcctat gtccagcgtg
     3781 aagtggctgc tgcctaatgg cacagtgctg agccacgcca gcagacaccc tagaatcagc
     3841 gtgctgaacg acggcaccct gaacttctct cacgtgctgc tgagcgacac cggcgtgtac
     3901 acctgtatgg tcacaaacgt ggccggcaac agcaacgcca gcgcctacct gaatgtgtct
     3961 accgccgaac tgaataccag caactactcc ttcttcacca ccgtgaccgt ggaaaccaca
     4021 gagatcagcc ccgaggacac caccagaaag tacaagcctg tgcctaccac cagcaccggc
     4081 taccagccag cctacaccac aagcacaacc gtgctgatcc agaccaccag ggtgcccaaa
     4141 caggtggcag tgcctgctac cgacaccacc gataagatgc agaccagcct ggacgaagtg
     4201 atgaagacca ccaaaggcgc gccggaagat caagttgatc caagattgat tgatggcaag
     4261 ggcggatctc tggaggtgct gttccaggga ccaggcggaa gcggcaatcc cggcggcgat
     4321 ctggcccccc agatgctgcg cgagctgcag gagaccaatg ccgccctgca ggacgtgcgc
     4381 gagctgctgc gccaccgcgt gaaggagatc accttcctga agaacaccgt gatggagtgc
     4441 gacgcctgcg gtatgcagcc ctctagaggc tccggctcca tcatcccagt tgaggaggag
     4501 aacccggact tctggaaccg cgaggcagcc gaggccctgg gtgccgccaa gaagctgcag
     4561 cctgcacaga cagccgccaa gaacctcatc atcttcctgg gcgatgggat gggggtgtct
     4621 acggtgacag ctgccaggat cctaaaaggg cagaagaagg acaaactggg gcctgagata
     4681 cccctggcca tggaccgctt cccatatgtg gctctgtcca agacatacaa tgtagacaaa
     4741 catgtgccag acagtggagc cacagccacg gcctacctgt gcggggtcaa gggcaacttc
     4801 cagaccattg gcttgagtgc agccgcccgc tttaaccagt gcaacacgac acgcggcaac
     4861 gaggtcatct ccgtgatgaa tcgggccaag aaagcaggga agtcagtggg agtggtaacc
     4921 accacacgag tgcagcacgc ctcgccagcc ggcacctacg cccacacggt gaaccgcaac
     4981 tggtactcgg acgccgacgt gcctgcctcg gcccgccagg aggggtgcca ggacatcgct
     5041 acgcagctca tctccaacat ggacattgac gtgatcctag gtggaggccg aaagtacatg
     5101 tttcgcatgg gaaccccaga ccctgagtac ccagatgact acagccaagg tgggaccagg
     5161 ctggacggga agaatctggt gcaggaatgg ctggcgaagc gccagggtgc ccggtatgtg
     5221 tggaaccgca ctgagctcat gcaggcttcc ctggacccgt ctgtgaccca tctcatgggt
     5281 ctctttgagc ctggagacat gaaatacgag atccaccgag actccacact ggacccctcc
     5341 ctgatggaga tgacagaggc tgccctgcgc ctgctgagca ggaacccccg cggcttcttc
     5401 ctcttcgtgg agggtggtcg catcgaccat ggtcatcatg aaagcagggc ttaccgggca
     5461 ctgactgaga cgatcatgtt cgacgacgcc attgagaggg cgggccagct caccagcgag
     5521 gaggacacgc tgagcctcgt cactgccgac cactcccacg tcttctcctt cggaggctac
     5581 cccctgcgag ggagctccat cttcgggctg gcccctggca aggcccggga caggaaggcc
     5641 tacacggtcc tcctatacgg aaacggtcca ggctatgtgc tcaaggacgg cgcccggccg
     5701 gatgttaccg agagcgagag cgggagcccc gagtatcggc agcagtcagc agtgcccctg
     5761 gacgaagaga cccacgcagg cgaggacgtg gcggtgttcg cgcgcggccc gcaggcgcac
     5821 ctggttcacg gcgtgcagga gcagaccttc atagcgcacg tcatggcctt cgccgcctgc
     5881 ctggagccct acaccgcctg cgacctggcg ccccccgccg gcaccaccga cgccgcgcac
     5941 ccgggtggcg gaggactgaa cgacatcttc gaggctcaga aaatcgaatg gcacgaacat
     6001 caccatcacc atcaccatca ccattaaggc cggcctgtag tcactggcac aacgcgttac
     6061 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc
     6121 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct
     6181 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6241 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct
     6301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6361 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     6421 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6481 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6541 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6601 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6661 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     6721 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     6781 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     6841 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     6901 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     6961 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     7021 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     7081 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     7141 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     7201 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     7261 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     7321 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     7381 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     7441 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7501 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7561 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     7621 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7681 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     7741 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7801 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7861 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     7921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     8041 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     8101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8161 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     8221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8341 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8581 aagggcgaca cggaaatgtt gaatactcat attcttcctt
//
