LOCUS       sequence-304031-        8695 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 8695)
  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 8695)
  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-LRFN2-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8695
                     /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             4300..4335
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4345..4368
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6028..6072
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6073..6099
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6239..6256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6245..6356
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6474..6494)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6479..6808
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6659..6794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6721..6740
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6856..6903
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6922..7521
                     /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(6922..6941)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7418..7438
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7651..7772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7688..7707)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7742..7761
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7825..8616,8617..8685))
                     /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     8448..8467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8686..8695,1..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 cggccgcctg tcctaagtac
     2761 tgcgtgtgcc agaatctgag cgagagcctg ggcacactgt gtccttctaa gggcctgctg
     2821 ttcgtgcctc ctgacatcga cagacggacc gtggaactga gactcggcgg caacttcatc
     2881 atccacatca gcagacagga cttcgccaac atgaccggcc tggtggatct gaccctgagc
     2941 agaaacacca tcagccatat ccagcctttc agcttcctgg acctggaaag cctgagaagc
     3001 ctgcacctgg acagcaacag actgcctagc ctgggcgaag ataccctgag aggcctggtc
     3061 aatctgcagc acctgatcgt gaacaacaac cagctcggcg gaatcgccga tgaggccttc
     3121 gaggattttc tgctgaccct ggaagatctg gacctgagct acaacaacct gcacggcctg
     3181 ccttgggata gcgtgcggag aatggttaac ctgcaccagc tgagcctgga ccacaacctg
     3241 ctggatcaca ttgccgaggg caccttcgcc gatctgcaga aactggccag actggacctc
     3301 acctccaacc ggctgcaaaa gctgcctcca gatccaatct tcgccagaag ccaggccagc
     3361 gctctgaccg ctacaccttt tgctcctcca ctgagcttca gctttggcgg caatcccctg
     3421 cactgcaact gcgaactgct gtggctgcgg agactggaaa gagatgacga tctggaaacc
     3481 tgcggctctc ctggcggact gaagggcaga tacttctggc acgtccgcga agaggaattc
     3541 gtgtgcgagc ctcctctgat cacccagcac acacacaagc tgctggtgct ggaaggacag
     3601 gccgccacac tgaagtgtaa agccatcggc gacccctctc cactgatcca ttgggtcgcc
     3661 cctgacgata gactcgtggg caatagcagc agaaccgccg tgtacgacaa cggcaccctg
     3721 gacatcttca tcaccaccag ccaggacagc ggcgccttta catgtatcgc cgctaatgcc
     3781 gctggcgagg ccacagctat ggtggaagtg tctatcgtgc agctgcccca cctgagcaac
     3841 agcaccagta gaacagcccc tccaaagagc cggctgagcg atatcacagg cagcagcaag
     3901 acaagcagag gcggcggagg atctggtggc ggagaacctc ctaagtctcc tccagaaaga
     3961 gccgtgctcg tcagcgaagt gaccacaaca tctgccctgg tcaagtggtc cgtgtccaag
     4021 tctgccccta gagtgaagat gtatcagctg cagtacaact gcagcgacga cgaggtgctg
     4081 atctacagaa tgatccccgc cagcaacaag gccttcgtgg tcaacaatct ggtgtccggc
     4141 accggctacg atctgtgcgt tctggccatg tgggacgaca ccgccacaac actgaccgcc
     4201 accaatatcg tgggctgcgc ccagttcttc accaaggccg attatcccca gtgccagagc
     4261 atgcactctc agatcctcgg aggcaccatg ggcgcgccgg aagatcaagt tgatccaaga
     4321 ttgattgatg gcaagggcgg atctctggag gtgctgttcc agggaccagg cggaagcggc
     4381 aatcccggcg gcgatctggc cccccagatg ctgcgcgagc tgcaggagac caatgccgcc
     4441 ctgcaggacg tgcgcgagct gctgcgccac cgcgtgaagg agatcacctt cctgaagaac
     4501 accgtgatgg agtgcgacgc ctgcggtatg cagccctcta gaggctccgg ctccatcatc
     4561 ccagttgagg aggagaaccc ggacttctgg aaccgcgagg cagccgaggc cctgggtgcc
     4621 gccaagaagc tgcagcctgc acagacagcc gccaagaacc tcatcatctt cctgggcgat
     4681 gggatggggg tgtctacggt gacagctgcc aggatcctaa aagggcagaa gaaggacaaa
     4741 ctggggcctg agatacccct ggccatggac cgcttcccat atgtggctct gtccaagaca
     4801 tacaatgtag acaaacatgt gccagacagt ggagccacag ccacggccta cctgtgcggg
     4861 gtcaagggca acttccagac cattggcttg agtgcagccg cccgctttaa ccagtgcaac
     4921 acgacacgcg gcaacgaggt catctccgtg atgaatcggg ccaagaaagc agggaagtca
     4981 gtgggagtgg taaccaccac acgagtgcag cacgcctcgc cagccggcac ctacgcccac
     5041 acggtgaacc gcaactggta ctcggacgcc gacgtgcctg cctcggcccg ccaggagggg
     5101 tgccaggaca tcgctacgca gctcatctcc aacatggaca ttgacgtgat cctaggtgga
     5161 ggccgaaagt acatgtttcg catgggaacc ccagaccctg agtacccaga tgactacagc
     5221 caaggtggga ccaggctgga cgggaagaat ctggtgcagg aatggctggc gaagcgccag
     5281 ggtgcccggt atgtgtggaa ccgcactgag ctcatgcagg cttccctgga cccgtctgtg
     5341 acccatctca tgggtctctt tgagcctgga gacatgaaat acgagatcca ccgagactcc
     5401 acactggacc cctccctgat ggagatgaca gaggctgccc tgcgcctgct gagcaggaac
     5461 ccccgcggct tcttcctctt cgtggagggt ggtcgcatcg accatggtca tcatgaaagc
     5521 agggcttacc gggcactgac tgagacgatc atgttcgacg acgccattga gagggcgggc
     5581 cagctcacca gcgaggagga cacgctgagc ctcgtcactg ccgaccactc ccacgtcttc
     5641 tccttcggag gctaccccct gcgagggagc tccatcttcg ggctggcccc tggcaaggcc
     5701 cgggacagga aggcctacac ggtcctccta tacggaaacg gtccaggcta tgtgctcaag
     5761 gacggcgccc ggccggatgt taccgagagc gagagcggga gccccgagta tcggcagcag
     5821 tcagcagtgc ccctggacga agagacccac gcaggcgagg acgtggcggt gttcgcgcgc
     5881 ggcccgcagg cgcacctggt tcacggcgtg caggagcaga ccttcatagc gcacgtcatg
     5941 gccttcgccg cctgcctgga gccctacacc gcctgcgacc tggcgccccc cgccggcacc
     6001 accgacgccg cgcacccggg tggcggagga ctgaacgaca tcttcgaggc tcagaaaatc
     6061 gaatggcacg aacatcacca tcaccatcac catcaccatt aaggccggcc tgtagtcact
     6121 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg
     6181 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc
     6241 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     6301 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa
     6361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     6421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     6481 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     6541 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     6601 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6661 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6721 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     6781 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     6841 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     6901 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     6961 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     7021 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     7081 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     7141 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     7201 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     7261 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     7321 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     7381 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg
     7441 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     7501 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat
     7561 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     7621 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     7681 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     7741 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     7801 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc
     7861 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7921 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     7981 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     8041 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     8101 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     8161 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     8221 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     8281 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     8341 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8401 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8461 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8521 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8581 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8641 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt
//
