LOCUS       sequence-474339-        7816 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 7816)
  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 7816)
  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-BTNL8-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7816
                     /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             3076..3087
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3421..3456
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             3466..3489
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5149..5193
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5194..5220
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5360..5377)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5366..5477
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5595..5615)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5600..5929
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5780..5915
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5842..5861
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5977..6024
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6043..6642
                     /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(6043..6062)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6539..6559
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6772..6893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6809..6828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6863..6882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6946..7737,7738..7806))
                     /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     7569..7588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7807..7816,1..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 atggcaagtg
     2761 tttggacctg ataagcctgt gcaggccctc gtgggagaag atgccgcctt tagctgcttt
     2821 ctgagcccca agacaaacgc cgaagccatg gaagtgcggt tcttcagagg ccagtttagc
     2881 agcgtggtgc acctgtacag agatggcaag gaccagcctt tcatgcagat gccccagtac
     2941 cagggcagaa caaagctggt caaggactct atcgccgagg gcagaatcag cctgcggctg
     3001 gaaaacatca cagtgctgga tgccggcctg tacggctgta gaatcagcag ccagagctac
     3061 taccagaagg ccatctggga gctgcaagtg tctgctctgg gaagcgtgcc actgatcagc
     3121 atcacaggct acgtggacag agacatccag ctgctgtgtc agagcagcgg ctggtttcct
     3181 agacctaccg ccaagtggaa gggccctcag ggccaagatc tgagcaccga cagccggacc
     3241 aatagagaca tgcacggcct gttcgacgtg gaaatctccc tgaccgtgca agagaatgcc
     3301 ggcagcatca gctgtagcat gagacacgcc cacctgagca gagaagtgga aagcagagtg
     3361 cagatcggcg acaccttctt cgagcctatt agctggcacc tggccaccaa aggcgcgccg
     3421 gaagatcaag ttgatccaag attgattgat ggcaagggcg gatctctgga ggtgctgttc
     3481 cagggaccag gcggaagcgg caatcccggc ggcgatctgg ccccccagat gctgcgcgag
     3541 ctgcaggaga ccaatgccgc cctgcaggac gtgcgcgagc tgctgcgcca ccgcgtgaag
     3601 gagatcacct tcctgaagaa caccgtgatg gagtgcgacg cctgcggtat gcagccctct
     3661 agaggctccg gctccatcat cccagttgag gaggagaacc cggacttctg gaaccgcgag
     3721 gcagccgagg ccctgggtgc cgccaagaag ctgcagcctg cacagacagc cgccaagaac
     3781 ctcatcatct tcctgggcga tgggatgggg gtgtctacgg tgacagctgc caggatccta
     3841 aaagggcaga agaaggacaa actggggcct gagatacccc tggccatgga ccgcttccca
     3901 tatgtggctc tgtccaagac atacaatgta gacaaacatg tgccagacag tggagccaca
     3961 gccacggcct acctgtgcgg ggtcaagggc aacttccaga ccattggctt gagtgcagcc
     4021 gcccgcttta accagtgcaa cacgacacgc ggcaacgagg tcatctccgt gatgaatcgg
     4081 gccaagaaag cagggaagtc agtgggagtg gtaaccacca cacgagtgca gcacgcctcg
     4141 ccagccggca cctacgccca cacggtgaac cgcaactggt actcggacgc cgacgtgcct
     4201 gcctcggccc gccaggaggg gtgccaggac atcgctacgc agctcatctc caacatggac
     4261 attgacgtga tcctaggtgg aggccgaaag tacatgtttc gcatgggaac cccagaccct
     4321 gagtacccag atgactacag ccaaggtggg accaggctgg acgggaagaa tctggtgcag
     4381 gaatggctgg cgaagcgcca gggtgcccgg tatgtgtgga accgcactga gctcatgcag
     4441 gcttccctgg acccgtctgt gacccatctc atgggtctct ttgagcctgg agacatgaaa
     4501 tacgagatcc accgagactc cacactggac ccctccctga tggagatgac agaggctgcc
     4561 ctgcgcctgc tgagcaggaa cccccgcggc ttcttcctct tcgtggaggg tggtcgcatc
     4621 gaccatggtc atcatgaaag cagggcttac cgggcactga ctgagacgat catgttcgac
     4681 gacgccattg agagggcggg ccagctcacc agcgaggagg acacgctgag cctcgtcact
     4741 gccgaccact cccacgtctt ctccttcgga ggctaccccc tgcgagggag ctccatcttc
     4801 gggctggccc ctggcaaggc ccgggacagg aaggcctaca cggtcctcct atacggaaac
     4861 ggtccaggct atgtgctcaa ggacggcgcc cggccggatg ttaccgagag cgagagcggg
     4921 agccccgagt atcggcagca gtcagcagtg cccctggacg aagagaccca cgcaggcgag
     4981 gacgtggcgg tgttcgcgcg cggcccgcag gcgcacctgg ttcacggcgt gcaggagcag
     5041 accttcatag cgcacgtcat ggccttcgcc gcctgcctgg agccctacac cgcctgcgac
     5101 ctggcgcccc ccgccggcac caccgacgcc gcgcacccgg gtggcggagg actgaacgac
     5161 atcttcgagg ctcagaaaat cgaatggcac gaacatcacc atcaccatca ccatcaccat
     5221 taaggccggc ctgtagtcac tggcacaacg cgttacccgg taagccaatc gggtatacac
     5281 ggtcgtcata ctgcagacag ggttcttcta ctttgcaaga tagtcttgag tagtaaaata
     5341 aatagataga gaaaatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5401 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5461 aatgaggaaa ttgcatcaca acactcaacc ctatctcggt ctattctttt gatttataag
     5521 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5581 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     5641 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     5701 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     5761 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     5821 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     5881 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     5941 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     6001 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgaccga gtacaagccc
     6061 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     6121 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     6181 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     6241 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     6301 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     6361 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     6421 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     6481 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacatc cgcgccccgc
     6541 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     6601 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacacgtgct acgagatttc
     6661 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     6721 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     6781 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     6841 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     6901 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcattacc aatgcttaat
     6961 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7021 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagcg ctgcgatgat
     7081 accgcgagaa ccacgctcac cggctccgga tttatcagca ataaaccagc cagccggaag
     7141 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7201 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccatcgc
     7261 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7321 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7381 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7441 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7501 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7561 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7621 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7681 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7741 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7801 actcatattc ttcctt
//
