LOCUS       sequence-495584-        7825 bp    DNA     circular SYN 17-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 7825)
  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 7825)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 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-KIR2DS2-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7825
                     /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             3430..3465
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             3475..3498
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5158..5202
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5203..5229
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5369..5386)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5375..5486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5604..5624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5609..5938
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5789..5924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5851..5870
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5986..6033
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6052..6651
                     /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(6052..6071)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6548..6568
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6781..6902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6818..6837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6872..6891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6955..7746,7747..7815))
                     /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     7578..7597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7816..7825,1..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 tgaaggcgtg
     2761 cacagaaaac cttctctgct ggcccatcct ggacctctgg tcaagagcga agagacagtg
     2821 atcctgcagt gttggagcga cgtccgcttc gagcactttc tgctgcacag agagggcaag
     2881 tacaaggaca cactgcacct gatcggagag caccacgatg gcgtgtccaa ggccaatttc
     2941 agcatcggcc ccatgatgca ggatctggcc ggcacataca gatgctacgg ctctgtgaca
     3001 cacagcccct atcagctgtc tgcccctagc gatcccctgg atatcgtgat caccggcctg
     3061 tacgagaagc ctagcctgtc tgctcagcct ggacctacag ttctggctgg cgaaagcgtg
     3121 accctgagct gtagcagcag atccagctac gacatgtacc acctgtccag agaaggcgag
     3181 gcccacgaga gaagattcag cgccggacct aaagtgaacg gcaccttcca ggccgatttt
     3241 ccactgggac ctgccacaca cggcggcacc tatagatgct ttggcagctt cagagacagc
     3301 ccttacgagt ggtccaacag cagcgatcct ctgctggtgt ctgtgaccgg caatcccagc
     3361 aatagctggc cttctccaac cgagcctagc agcaagacag gcaaccccag acatctgcat
     3421 ggcgcgccgg aagatcaagt tgatccaaga ttgattgatg gcaagggcgg atctctggag
     3481 gtgctgttcc agggaccagg cggaagcggc aatcccggcg gcgatctggc cccccagatg
     3541 ctgcgcgagc tgcaggagac caatgccgcc ctgcaggacg tgcgcgagct gctgcgccac
     3601 cgcgtgaagg agatcacctt cctgaagaac accgtgatgg agtgcgacgc ctgcggtatg
     3661 cagccctcta gaggctccgg ctccatcatc ccagttgagg aggagaaccc ggacttctgg
     3721 aaccgcgagg cagccgaggc cctgggtgcc gccaagaagc tgcagcctgc acagacagcc
     3781 gccaagaacc tcatcatctt cctgggcgat gggatggggg tgtctacggt gacagctgcc
     3841 aggatcctaa aagggcagaa gaaggacaaa ctggggcctg agatacccct ggccatggac
     3901 cgcttcccat atgtggctct gtccaagaca tacaatgtag acaaacatgt gccagacagt
     3961 ggagccacag ccacggccta cctgtgcggg gtcaagggca acttccagac cattggcttg
     4021 agtgcagccg cccgctttaa ccagtgcaac acgacacgcg gcaacgaggt catctccgtg
     4081 atgaatcggg ccaagaaagc agggaagtca gtgggagtgg taaccaccac acgagtgcag
     4141 cacgcctcgc cagccggcac ctacgcccac acggtgaacc gcaactggta ctcggacgcc
     4201 gacgtgcctg cctcggcccg ccaggagggg tgccaggaca tcgctacgca gctcatctcc
     4261 aacatggaca ttgacgtgat cctaggtgga ggccgaaagt acatgtttcg catgggaacc
     4321 ccagaccctg agtacccaga tgactacagc caaggtggga ccaggctgga cgggaagaat
     4381 ctggtgcagg aatggctggc gaagcgccag ggtgcccggt atgtgtggaa ccgcactgag
     4441 ctcatgcagg cttccctgga cccgtctgtg acccatctca tgggtctctt tgagcctgga
     4501 gacatgaaat acgagatcca ccgagactcc acactggacc cctccctgat ggagatgaca
     4561 gaggctgccc tgcgcctgct gagcaggaac ccccgcggct tcttcctctt cgtggagggt
     4621 ggtcgcatcg accatggtca tcatgaaagc agggcttacc gggcactgac tgagacgatc
     4681 atgttcgacg acgccattga gagggcgggc cagctcacca gcgaggagga cacgctgagc
     4741 ctcgtcactg ccgaccactc ccacgtcttc tccttcggag gctaccccct gcgagggagc
     4801 tccatcttcg ggctggcccc tggcaaggcc cgggacagga aggcctacac ggtcctccta
     4861 tacggaaacg gtccaggcta tgtgctcaag gacggcgccc ggccggatgt taccgagagc
     4921 gagagcggga gccccgagta tcggcagcag tcagcagtgc ccctggacga agagacccac
     4981 gcaggcgagg acgtggcggt gttcgcgcgc ggcccgcagg cgcacctggt tcacggcgtg
     5041 caggagcaga ccttcatagc gcacgtcatg gccttcgccg cctgcctgga gccctacacc
     5101 gcctgcgacc tggcgccccc cgccggcacc accgacgccg cgcacccggg tggcggagga
     5161 ctgaacgaca tcttcgaggc tcagaaaatc gaatggcacg aacatcacca tcaccatcac
     5221 catcaccatt aaggccggcc tgtagtcact ggcacaacgc gttacccggt aagccaatcg
     5281 ggtatacacg gtcgtcatac tgcagacagg gttcttctac tttgcaagat agtcttgagt
     5341 agtaaaataa atagatagag aaaatcagcc tcgactgtgc cttctagttg ccagccatct
     5401 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5461 tcctaataaa atgaggaaat tgcatcacaa cactcaaccc tatctcggtc tattcttttg
     5521 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5581 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     5641 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     5701 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5761 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     5821 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     5881 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     5941 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     6001 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catgaccgag
     6061 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6121 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6181 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     6241 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     6301 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     6361 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6421 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     6481 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacatcc
     6541 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     6601 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acacgtgcta
     6661 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     6721 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     6781 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     6841 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     6901 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcattacca
     6961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc
     7081 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc
     7141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7261 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7801 atgttgaata ctcatattct tcctt
//
