LOCUS       sequence-303845-        8236 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 8236)
  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 8236)
  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-TNFRSF8-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8236
                     /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             3841..3876
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             3886..3909
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5569..5613
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5614..5640
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5780..5797)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5786..5897
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6015..6035)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6020..6349
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6200..6335
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6262..6281
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6397..6444
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6463..7062
                     /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(6463..6482)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6959..6979
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7192..7313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7229..7248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7283..7302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7366..8157,8158..8226))
                     /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     7989..8008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8227..8236,1..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 cggccgcctt tccacaagac
     2761 agacccttcg aggatacctg ccacggcaac cccagccact actacgataa ggccgtgcgg
     2821 agatgctgct acagatgccc catgggactg ttccccacac agcagtgccc tcagaggcct
     2881 accgattgca gaaagcagtg cgagcccgac tactacctgg acgaggccga tagatgtacc
     2941 gcctgcgtga cctgcagcag agatgacctg gtggaaaaga ccccttgcgc ctggaacagc
     3001 agcagagtgt gcgagtgtag acccggcatg ttctgtagca ccagcgccgt gaatagctgc
     3061 gccagatgct tcttccacag cgtgtgtcct gccggcatga tcgtgaagtt tcccggcaca
     3121 gcccagaaaa acaccgtgtg cgaacctgcc tctcctggcg ttagtcctgc ctgtgcctct
     3181 ccagagaact gcaaagagcc tagcagcggc acaatccctc aggccaagcc tacacctgtg
     3241 tctccagcca caagcagcgc cagcacaatg cctgttagag gcggaaccag actggcccaa
     3301 gaagccgcca gcaagctgac aagagcccct gatagcccta gctccgtggg cagacctagc
     3361 tctgatcctg gcctgtctcc tacacagccc tgtcctgaag gcagcggcga ctgtaggaaa
     3421 cagtgcgaac cagattacta tctcgatgag gccggcagat gcacagcctg tgtgtcttgt
     3481 agcagggacg acctggtcga gaaaactccc tgtgcttgga acagctcccg gacctgcgaa
     3541 tgcagacctg gaatgatctg tgccacctcc gccaccaact cttgtgccag atgcgtgccc
     3601 tatcctatct gtgccgccga gacagtgacc aagcctcagg atatggccga gaaggacacc
     3661 acctttgagg cccctccact gggcacacag cctgactgta atcccacacc tgagaacggc
     3721 gaagcccctg cctctacaag ccctacacag agcctgctgg tggatagcca ggccagcaag
     3781 accctgccta ttcctacatc tgcccctgtg gctctgagca gcacaggcaa aggcgcgccg
     3841 gaagatcaag ttgatccaag attgattgat ggcaagggcg gatctctgga ggtgctgttc
     3901 cagggaccag gcggaagcgg caatcccggc ggcgatctgg ccccccagat gctgcgcgag
     3961 ctgcaggaga ccaatgccgc cctgcaggac gtgcgcgagc tgctgcgcca ccgcgtgaag
     4021 gagatcacct tcctgaagaa caccgtgatg gagtgcgacg cctgcggtat gcagccctct
     4081 agaggctccg gctccatcat cccagttgag gaggagaacc cggacttctg gaaccgcgag
     4141 gcagccgagg ccctgggtgc cgccaagaag ctgcagcctg cacagacagc cgccaagaac
     4201 ctcatcatct tcctgggcga tgggatgggg gtgtctacgg tgacagctgc caggatccta
     4261 aaagggcaga agaaggacaa actggggcct gagatacccc tggccatgga ccgcttccca
     4321 tatgtggctc tgtccaagac atacaatgta gacaaacatg tgccagacag tggagccaca
     4381 gccacggcct acctgtgcgg ggtcaagggc aacttccaga ccattggctt gagtgcagcc
     4441 gcccgcttta accagtgcaa cacgacacgc ggcaacgagg tcatctccgt gatgaatcgg
     4501 gccaagaaag cagggaagtc agtgggagtg gtaaccacca cacgagtgca gcacgcctcg
     4561 ccagccggca cctacgccca cacggtgaac cgcaactggt actcggacgc cgacgtgcct
     4621 gcctcggccc gccaggaggg gtgccaggac atcgctacgc agctcatctc caacatggac
     4681 attgacgtga tcctaggtgg aggccgaaag tacatgtttc gcatgggaac cccagaccct
     4741 gagtacccag atgactacag ccaaggtggg accaggctgg acgggaagaa tctggtgcag
     4801 gaatggctgg cgaagcgcca gggtgcccgg tatgtgtgga accgcactga gctcatgcag
     4861 gcttccctgg acccgtctgt gacccatctc atgggtctct ttgagcctgg agacatgaaa
     4921 tacgagatcc accgagactc cacactggac ccctccctga tggagatgac agaggctgcc
     4981 ctgcgcctgc tgagcaggaa cccccgcggc ttcttcctct tcgtggaggg tggtcgcatc
     5041 gaccatggtc atcatgaaag cagggcttac cgggcactga ctgagacgat catgttcgac
     5101 gacgccattg agagggcggg ccagctcacc agcgaggagg acacgctgag cctcgtcact
     5161 gccgaccact cccacgtctt ctccttcgga ggctaccccc tgcgagggag ctccatcttc
     5221 gggctggccc ctggcaaggc ccgggacagg aaggcctaca cggtcctcct atacggaaac
     5281 ggtccaggct atgtgctcaa ggacggcgcc cggccggatg ttaccgagag cgagagcggg
     5341 agccccgagt atcggcagca gtcagcagtg cccctggacg aagagaccca cgcaggcgag
     5401 gacgtggcgg tgttcgcgcg cggcccgcag gcgcacctgg ttcacggcgt gcaggagcag
     5461 accttcatag cgcacgtcat ggccttcgcc gcctgcctgg agccctacac cgcctgcgac
     5521 ctggcgcccc ccgccggcac caccgacgcc gcgcacccgg gtggcggagg actgaacgac
     5581 atcttcgagg ctcagaaaat cgaatggcac gaacatcacc atcaccatca ccatcaccat
     5641 taaggccggc ctgtagtcac tggcacaacg cgttacccgg taagccaatc gggtatacac
     5701 ggtcgtcata ctgcagacag ggttcttcta ctttgcaaga tagtcttgag tagtaaaata
     5761 aatagataga gaaaatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5821 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5881 aatgaggaaa ttgcatcaca acactcaacc ctatctcggt ctattctttt gatttataag
     5941 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6001 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     6061 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     6121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     6181 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     6241 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     6301 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     6361 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     6421 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgaccga gtacaagccc
     6481 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     6541 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     6601 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     6661 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     6721 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     6781 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     6841 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     6901 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacatc cgcgccccgc
     6961 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     7021 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacacgtgct acgagatttc
     7081 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     7141 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     7201 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     7261 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     7321 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcattacc aatgcttaat
     7381 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7441 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagcg ctgcgatgat
     7501 accgcgagaa ccacgctcac cggctccgga tttatcagca ataaaccagc cagccggaag
     7561 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7621 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccatcgc
     7681 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7741 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7801 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7861 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7921 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7981 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8041 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8101 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8161 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8221 actcatattc ttcctt
//
