LOCUS       sequence-304065-        8842 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 8842)
  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 8842)
  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-LRIT3-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8842
                     /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     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             4447..4482
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4492..4515
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6175..6219
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6220..6246
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6386..6403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6392..6503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6621..6641)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6626..6955
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6806..6941
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6868..6887
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7003..7050
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7069..7668
                     /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(7069..7088)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7565..7585
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7798..7919
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7835..7854)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7889..7908
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7972..8763,8764..8832))
                     /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     8595..8614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8833..8842,1..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 gtgtcctagc
     2761 cagtgtacat gtgactacca cggcagaaac gacggctctg gaagcagact ggtgctgtgc
     2821 aacgacatgg acatgaacga gctgcccacc aacctgcctg tggacaccgt gaagctgcgg
     2881 atcgagaaaa ccgtgatcag aagaatcagc gccgaggcct tctactacct ggtggaactg
     2941 cagtacctgt gggtcaccta caacagcgtg gccagcatcg accccagcag cttctacaat
     3001 ctgaagcagc tgcacgagct gcggctggat ggaaattctc tggctgcctt tccttgggcc
     3061 tctctgctgg atatgcctct gctgagaacc ctggacctgc acaacaacaa gatcaccagc
     3121 gtgcccaacg aggccctgag atacctgaag aacctggcct acctggatct gagcagcaac
     3181 agactgacaa ccctgcctcc tgacttcctg gaaagctgga cccacctggt gtctacacct
     3241 agcggagtgc tggatctgtc ccctagcaga atcatcctgg gcctgcagga caacccctgg
     3301 ttctgcgatt gccacatcag caagatgatc gagctgagca aggtggtgga ccctgctatt
     3361 gtgctgctgg accctctgat gacctgcagc gagcctgaga gactgaccgg cattctgttc
     3421 cagagagccg agctggaaca ctgcctgaag cctagcgtga tgaccagcgc cacaaagatc
     3481 atgagcgccc tgggctctaa cgtgctgctg agatgtgatg ccaccggctt tcccacacct
     3541 cagatcacct ggaccagatc cgacagcagc cccgtgaact acaccgtgat ccaagagtct
     3601 cccgaagagg gcgtcagatg gtccattatg agcctgacag gcatcagcag caaggacgcc
     3661 ggcgattaca agtgcaaggc caagaatctg gccggcatgt ctgaggccgt ggtcacagtg
     3721 acagtgctgg gcatcaccac cacacctatt cctccagaca ccagcgagag aaccggcgat
     3781 catcctgaat gggatgtgca gcctggcagc ggcagatcta catctgtgtc tagcgccagc
     3841 tcctacctgt ggtctagcag ctttagcccc acctccagct tttccgccag cacactgtct
     3901 ccaccaagta ccgcaagctt cagtctgagc cctttcagca gcagcaccgt gtcctctacc
     3961 acaacactga gcaccagcat cagcgccagc accaccatgg ccaacaagag aagctttcag
     4021 ctgcaccaag gcggcaagag aaacctgaag gtggccaaga acggcagcaa gctgcctcca
     4081 gccagcacct ccaaaaaaga ggaactggcc ctgctggatc agaccatgct gacagagaca
     4141 aacgccgcca tcgagaacct gagagtggtg tccgagacaa aagaaagcgt gaccctgacc
     4201 tggaacatga tcaacaccac acacaacagc gccgtgaccg tgctgtactc taagtacggc
     4261 ggaaaggacc tgctgctcct gaacgccgac agctccaaga atcaagtgac catcgacgga
     4321 ctggaacctg gcggccagta tatggcctgt gtgtgtccta aaggcgtgcc acctcagaaa
     4381 gaccagtgca tcaccttcag caccgagaga gtggaaggcg acgacagcca gtggtctggc
     4441 gcgccggaag atcaagttga tccaagattg attgatggca agggcggatc tctggaggtg
     4501 ctgttccagg gaccaggcgg aagcggcaat cccggcggcg atctggcccc ccagatgctg
     4561 cgcgagctgc aggagaccaa tgccgccctg caggacgtgc gcgagctgct gcgccaccgc
     4621 gtgaaggaga tcaccttcct gaagaacacc gtgatggagt gcgacgcctg cggtatgcag
     4681 ccctctagag gctccggctc catcatccca gttgaggagg agaacccgga cttctggaac
     4741 cgcgaggcag ccgaggccct gggtgccgcc aagaagctgc agcctgcaca gacagccgcc
     4801 aagaacctca tcatcttcct gggcgatggg atgggggtgt ctacggtgac agctgccagg
     4861 atcctaaaag ggcagaagaa ggacaaactg gggcctgaga tacccctggc catggaccgc
     4921 ttcccatatg tggctctgtc caagacatac aatgtagaca aacatgtgcc agacagtgga
     4981 gccacagcca cggcctacct gtgcggggtc aagggcaact tccagaccat tggcttgagt
     5041 gcagccgccc gctttaacca gtgcaacacg acacgcggca acgaggtcat ctccgtgatg
     5101 aatcgggcca agaaagcagg gaagtcagtg ggagtggtaa ccaccacacg agtgcagcac
     5161 gcctcgccag ccggcaccta cgcccacacg gtgaaccgca actggtactc ggacgccgac
     5221 gtgcctgcct cggcccgcca ggaggggtgc caggacatcg ctacgcagct catctccaac
     5281 atggacattg acgtgatcct aggtggaggc cgaaagtaca tgtttcgcat gggaacccca
     5341 gaccctgagt acccagatga ctacagccaa ggtgggacca ggctggacgg gaagaatctg
     5401 gtgcaggaat ggctggcgaa gcgccagggt gcccggtatg tgtggaaccg cactgagctc
     5461 atgcaggctt ccctggaccc gtctgtgacc catctcatgg gtctctttga gcctggagac
     5521 atgaaatacg agatccaccg agactccaca ctggacccct ccctgatgga gatgacagag
     5581 gctgccctgc gcctgctgag caggaacccc cgcggcttct tcctcttcgt ggagggtggt
     5641 cgcatcgacc atggtcatca tgaaagcagg gcttaccggg cactgactga gacgatcatg
     5701 ttcgacgacg ccattgagag ggcgggccag ctcaccagcg aggaggacac gctgagcctc
     5761 gtcactgccg accactccca cgtcttctcc ttcggaggct accccctgcg agggagctcc
     5821 atcttcgggc tggcccctgg caaggcccgg gacaggaagg cctacacggt cctcctatac
     5881 ggaaacggtc caggctatgt gctcaaggac ggcgcccggc cggatgttac cgagagcgag
     5941 agcgggagcc ccgagtatcg gcagcagtca gcagtgcccc tggacgaaga gacccacgca
     6001 ggcgaggacg tggcggtgtt cgcgcgcggc ccgcaggcgc acctggttca cggcgtgcag
     6061 gagcagacct tcatagcgca cgtcatggcc ttcgccgcct gcctggagcc ctacaccgcc
     6121 tgcgacctgg cgccccccgc cggcaccacc gacgccgcgc acccgggtgg cggaggactg
     6181 aacgacatct tcgaggctca gaaaatcgaa tggcacgaac atcaccatca ccatcaccat
     6241 caccattaag gccggcctgt agtcactggc acaacgcgtt acccggtaag ccaatcgggt
     6301 atacacggtc gtcatactgc agacagggtt cttctacttt gcaagatagt cttgagtagt
     6361 aaaataaata gatagagaaa atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6421 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6481 taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat tcttttgatt
     6541 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6601 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     6661 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     6721 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6781 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6841 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     6901 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6961 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
     7021 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gaccgagtac
     7081 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     7141 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     7201 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     7261 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7321 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7381 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7441 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7501 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     7561 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7621 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga
     7681 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     7741 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     7801 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     7861 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7921 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc attaccaatg
     7981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     8041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     8101 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     8161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     8221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     8281 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8821 ttgaatactc atattcttcc tt
//
