LOCUS       sequence-493721-        8905 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 8905)
  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 8905)
  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-EDA2R-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8905
                     /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        3105..3484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3485..3688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3639..3659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3685..3709
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3919..3937
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3919..3936
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     3950..3969
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      4081..4090
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4510..4545
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4555..4578
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6238..6282
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6283..6309
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6449..6466)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6455..6566
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6684..6704)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6689..7018
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6869..7004
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6931..6950
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7066..7113
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7132..7731
                     /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(7132..7151)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7628..7648
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7861..7982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7898..7917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7952..7971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8035..8826,8827..8895))
                     /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     8658..8677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8896..8905,1..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 ctgagagatc ccctcataat ttccccaaag
     1741 cgtaaccatg tgtgaataaa ttttgagcta gtagggttgc agccacgagt aagtcttccc
     1801 ttgttattgt gtagccagaa tgccgcaaaa cttccatgcc taagcgaact gttgagagta
     1861 cgtttcgatt tctgactgtg ttagcctgga agtgcttgtc ccaaccttgt ttctgagcat
     1921 gaacgcccgc aagccaacat gttagttgaa gcatcagggc gattagcagc atgatatcaa
     1981 aacgctctga gctgctcgtt cggctatggc gtaggcctag tccgtaggca ggacttttca
     2041 agtctcggaa ggtttcttca atctgcattc gcttcgaata gatattaaca agttgtttgg
     2101 gtgttcgaat ttcaacaggt aagttagttg ctagaaccca tggctccttt gccgacgctg
     2161 agtagatttt aggtgacggg tggtgacaat gagtccgtgt cgagcgctga ttttttcggc
     2221 ctttagagcg agatttatac aatagaattt ggcatgagat tggattgctt ttagtcagcc
     2281 tcttatagcc taaagtcttt gagtgactag atgacatatc atgtaagttg ctgataggtt
     2341 tccagttttc cgctcctagg tctgcatatt gtacttttcc tcttactcga cttaaccagt
     2401 accaacccag cttctcaacg gatttatacc atggcacttt aaagccagca tcactgacaa
     2461 tgagcggtgt ggtgttactc ggtagaatgc tcgcaaggtc ggctagaaat tggtcatgag
     2521 ctttctttga acattgctct gaaagcggga acgctttctc ataaagagta acagaacgac
     2581 cgtgtagtgc gactgaagct cgcaatacca taagtcgttt ttgctcacga atatcagacc
     2641 agtcaacaag tacaatgggc atcgtattgc ccgaacagat aaagctagca tgccaacggt
     2701 atacagcgag tcgctctttg tggaggtgac gattacctaa caatcggtcg attcgtttga
     2761 tgttatgttt tgttctcgct ttggttggca ggttacggcc aagttcggta agagtgagag
     2821 ttttacagtc aagtaatgcg tggcaagcca acgttaagct gttgagtcgt tttaagtgta
     2881 attcggggca gaattggtaa agagagtcgt gtaaaatatc gagttcgcac atcttgttgt
     2941 ctgattattg atttttcgcg aaaccatttg atcatatgac aagatgtgta tccaccttaa
     3001 cttaatgatt tttaccaaaa tcattagggg attcatcagt acgttgtatc tatatcataa
     3061 tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac
     3121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     3541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     3601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     3661 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
     3721 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg
     3781 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag
     3841 agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt
     3901 atgtatcata cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc
     3961 tccacaggtg tccactccca ggtccaactg cacctcggtt ctatcgaaaa cgcgcctgct
     4021 agcgccacca tgaagaccat cattgccctg agctacatct tctgcctggt gttcgctgcg
     4081 gccgccatgg attgccaaga gaacgagtat tgggaccagt ggggcagatg cgtgacctgt
     4141 caaagatgtg gccctggcca agagctgagc aaggattgtg gatacggcga aggcggcgac
     4201 gcctactgta cagcttgtcc tcctcggaga tacaagagca gctggggaca ccacagatgc
     4261 cagagctgta tcacctgtgc cgtgatcaac cgggtgcaga aagtgaattg caccgccacc
     4321 tctaacgccg tgtgtggcga ttgcctgcct cggttctaca gaaagacaag aatcggcggc
     4381 ctgcaggacc aagagtgcat cccttgtacc aagcagaccc ctacctctga ggtgcagtgt
     4441 gcctttcagc tgagcctggt ggaagccgac acacctacag tgcctccaca agaggctaca
     4501 ggcgcgccgg aagatcaagt tgatccaaga ttgattgatg gcaagggcgg atctctggag
     4561 gtgctgttcc agggaccagg cggaagcggc aatcccggcg gcgatctggc cccccagatg
     4621 ctgcgcgagc tgcaggagac caatgccgcc ctgcaggacg tgcgcgagct gctgcgccac
     4681 cgcgtgaagg agatcacctt cctgaagaac accgtgatgg agtgcgacgc ctgcggtatg
     4741 cagccctcta gaggctccgg ctccatcatc ccagttgagg aggagaaccc ggacttctgg
     4801 aaccgcgagg cagccgaggc cctgggtgcc gccaagaagc tgcagcctgc acagacagcc
     4861 gccaagaacc tcatcatctt cctgggcgat gggatggggg tgtctacggt gacagctgcc
     4921 aggatcctaa aagggcagaa gaaggacaaa ctggggcctg agatacccct ggccatggac
     4981 cgcttcccat atgtggctct gtccaagaca tacaatgtag acaaacatgt gccagacagt
     5041 ggagccacag ccacggccta cctgtgcggg gtcaagggca acttccagac cattggcttg
     5101 agtgcagccg cccgctttaa ccagtgcaac acgacacgcg gcaacgaggt catctccgtg
     5161 atgaatcggg ccaagaaagc agggaagtca gtgggagtgg taaccaccac acgagtgcag
     5221 cacgcctcgc cagccggcac ctacgcccac acggtgaacc gcaactggta ctcggacgcc
     5281 gacgtgcctg cctcggcccg ccaggagggg tgccaggaca tcgctacgca gctcatctcc
     5341 aacatggaca ttgacgtgat cctaggtgga ggccgaaagt acatgtttcg catgggaacc
     5401 ccagaccctg agtacccaga tgactacagc caaggtggga ccaggctgga cgggaagaat
     5461 ctggtgcagg aatggctggc gaagcgccag ggtgcccggt atgtgtggaa ccgcactgag
     5521 ctcatgcagg cttccctgga cccgtctgtg acccatctca tgggtctctt tgagcctgga
     5581 gacatgaaat acgagatcca ccgagactcc acactggacc cctccctgat ggagatgaca
     5641 gaggctgccc tgcgcctgct gagcaggaac ccccgcggct tcttcctctt cgtggagggt
     5701 ggtcgcatcg accatggtca tcatgaaagc agggcttacc gggcactgac tgagacgatc
     5761 atgttcgacg acgccattga gagggcgggc cagctcacca gcgaggagga cacgctgagc
     5821 ctcgtcactg ccgaccactc ccacgtcttc tccttcggag gctaccccct gcgagggagc
     5881 tccatcttcg ggctggcccc tggcaaggcc cgggacagga aggcctacac ggtcctccta
     5941 tacggaaacg gtccaggcta tgtgctcaag gacggcgccc ggccggatgt taccgagagc
     6001 gagagcggga gccccgagta tcggcagcag tcagcagtgc ccctggacga agagacccac
     6061 gcaggcgagg acgtggcggt gttcgcgcgc ggcccgcagg cgcacctggt tcacggcgtg
     6121 caggagcaga ccttcatagc gcacgtcatg gccttcgccg cctgcctgga gccctacacc
     6181 gcctgcgacc tggcgccccc cgccggcacc accgacgccg cgcacccggg tggcggagga
     6241 ctgaacgaca tcttcgaggc tcagaaaatc gaatggcacg aacatcacca tcaccatcac
     6301 catcaccatt aaggccggcc tgtagtcact ggcacaacgc gttacccggt aagccaatcg
     6361 ggtatacacg gtcgtcatac tgcagacagg gttcttctac tttgcaagat agtcttgagt
     6421 agtaaaataa atagatagag aaaatcagcc tcgactgtgc cttctagttg ccagccatct
     6481 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6541 tcctaataaa atgaggaaat tgcatcacaa cactcaaccc tatctcggtc tattcttttg
     6601 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6661 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     6721 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     6781 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     6841 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     6901 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     6961 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     7021 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     7081 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catgaccgag
     7141 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     7201 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     7261 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     7321 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     7381 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     7441 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     7501 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     7561 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacatcc
     7621 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     7681 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acacgtgcta
     7741 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     7801 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     7861 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     7921 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     7981 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcattacca
     8041 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     8101 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc
     8161 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc
     8221 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     8281 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     8341 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     8401 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     8461 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     8521 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     8581 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     8641 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8701 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8761 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8821 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8881 atgttgaata ctcatattct tcctt
//
