LOCUS       sequence-304091-        8971 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 8971)
  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 8971)
  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-LRRN3-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8971
                     /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             4576..4611
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4621..4644
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6304..6348
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6349..6375
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6515..6532)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6521..6632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6750..6770)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6755..7084
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6935..7070
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6997..7016
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7132..7179
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7198..7797
                     /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(7198..7217)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7694..7714
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7927..8048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7964..7983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8018..8037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8101..8892,8893..8961))
                     /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     8724..8743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8962..8971,1..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 cggccgccgt ggataagaaa
     2761 gtggactgcc ctagactgtg tacctgcgag atcagaccct ggttcacccc tcggagcatc
     2821 tacatggaag ccagcaccgt ggactgcaac gatctgggcc tgctgacctt tcctgccaga
     2881 ctgcctgcca acacacagat cctgctgctg cagaccaaca atatcgccaa gatcgagtac
     2941 agcaccgact ttcccgtgaa cctgaccggc ctggatctga gccagaacaa cctgagcagc
     3001 gtgaccaaca tcaacgtgaa gaagatgccc cagctgctga gcgtgtacct ggaagagaac
     3061 aagctgaccg agctgcccga gaagtgtctg agcgagctga gcaacctgca agagctgtac
     3121 atcaaccaca acctgctgag cacaatcagc cctggcgcct ttatcggcct gcacaatctg
     3181 ctcagactgc acctgaacag caaccggctg cagatgatca acagcaagtg gttcgacgcc
     3241 ctgcctaacc tggaaatcct gatgatcggc gagaacccca tcatccggat caaggacatg
     3301 aacttcaagc ccctgatcaa cctgcggagc ctggttatcg ccggcatcaa cctgacagag
     3361 atccccgaca atgccctcgt gggcctcgag aacctggaat ccatcagctt ctacgacaac
     3421 cgcctgatca aggtgccaca cgtggccctg cagaaggtgg tcaacctgaa gttcctggac
     3481 ctgaacaaga accctatcaa cagaatcaga cggggcgact tcagcaacat gctgcatctg
     3541 aaagagctgg gcatcaacaa catgcccgag ctgatctcca tcgacagcct ggccgtcgac
     3601 aatctgcccg acctgagaaa gatcgaggcc accaacaatc cccggctgag ctacattcac
     3661 cccaacgcct ttttcagact gcccaagctg gaatccctga tgctgaatag caacgccctg
     3721 agcgccctgt accacggcac aattgagagc ctgccaaacc tgaaagaaat cagcatccac
     3781 agcaatccca tccgctgcga ctgcgtgatc cggtggatga acatgaacaa gaccaatatc
     3841 cggttcatgg aacccgatag cctgttctgc gtggaccctc ctgagttcca gggccagaat
     3901 gtgcgccagg tgcacttccg ggacatgatg gaaatctgcc tgcctctgat cgcccctgag
     3961 agcttcccca gcaacctgaa tgtggaagcc ggcagctacg tgtccttcca ctgtagagcc
     4021 acagccgagc ctcagcctga gatctactgg atcacaccca gcggccagaa gctgctgcct
     4081 aacacactga ccgacaagtt ctacgtgcac agcgagggca ccctggacat caatggcgtg
     4141 acccctaaag aaggcggcct gtacacctgt atcgccacca atcttgtggg cgccgacctg
     4201 aagtccgtga tgattaaggt ggacggcagc ttccctcagg acaacaacgg cagcctgaac
     4261 atcaagatca gagacatcca ggccaacagc gtgctggtgt cttggaaggc cagcagcaag
     4321 atcctgaagt ctagcgtgaa gtggaccgcc ttcgtgaaaa ccgagaacag ccacgccgct
     4381 cagagcgcca gaattccttc cgacgtgaag gtgtacaatc tgacccatct gaaccccagc
     4441 accgagtaca agatctgcat cgatatcccc accatctacc agaaaaaccg gaagaaatgc
     4501 gtgaacgtga ccaccaaggg actgcacccc gaccagaaag agtacgagaa gaacaacacc
     4561 accacaggcg cgccggaaga tcaagttgat ccaagattga ttgatggcaa gggcggatct
     4621 ctggaggtgc tgttccaggg accaggcgga agcggcaatc ccggcggcga tctggccccc
     4681 cagatgctgc gcgagctgca ggagaccaat gccgccctgc aggacgtgcg cgagctgctg
     4741 cgccaccgcg tgaaggagat caccttcctg aagaacaccg tgatggagtg cgacgcctgc
     4801 ggtatgcagc cctctagagg ctccggctcc atcatcccag ttgaggagga gaacccggac
     4861 ttctggaacc gcgaggcagc cgaggccctg ggtgccgcca agaagctgca gcctgcacag
     4921 acagccgcca agaacctcat catcttcctg ggcgatggga tgggggtgtc tacggtgaca
     4981 gctgccagga tcctaaaagg gcagaagaag gacaaactgg ggcctgagat acccctggcc
     5041 atggaccgct tcccatatgt ggctctgtcc aagacataca atgtagacaa acatgtgcca
     5101 gacagtggag ccacagccac ggcctacctg tgcggggtca agggcaactt ccagaccatt
     5161 ggcttgagtg cagccgcccg ctttaaccag tgcaacacga cacgcggcaa cgaggtcatc
     5221 tccgtgatga atcgggccaa gaaagcaggg aagtcagtgg gagtggtaac caccacacga
     5281 gtgcagcacg cctcgccagc cggcacctac gcccacacgg tgaaccgcaa ctggtactcg
     5341 gacgccgacg tgcctgcctc ggcccgccag gaggggtgcc aggacatcgc tacgcagctc
     5401 atctccaaca tggacattga cgtgatccta ggtggaggcc gaaagtacat gtttcgcatg
     5461 ggaaccccag accctgagta cccagatgac tacagccaag gtgggaccag gctggacggg
     5521 aagaatctgg tgcaggaatg gctggcgaag cgccagggtg cccggtatgt gtggaaccgc
     5581 actgagctca tgcaggcttc cctggacccg tctgtgaccc atctcatggg tctctttgag
     5641 cctggagaca tgaaatacga gatccaccga gactccacac tggacccctc cctgatggag
     5701 atgacagagg ctgccctgcg cctgctgagc aggaaccccc gcggcttctt cctcttcgtg
     5761 gagggtggtc gcatcgacca tggtcatcat gaaagcaggg cttaccgggc actgactgag
     5821 acgatcatgt tcgacgacgc cattgagagg gcgggccagc tcaccagcga ggaggacacg
     5881 ctgagcctcg tcactgccga ccactcccac gtcttctcct tcggaggcta ccccctgcga
     5941 gggagctcca tcttcgggct ggcccctggc aaggcccggg acaggaaggc ctacacggtc
     6001 ctcctatacg gaaacggtcc aggctatgtg ctcaaggacg gcgcccggcc ggatgttacc
     6061 gagagcgaga gcgggagccc cgagtatcgg cagcagtcag cagtgcccct ggacgaagag
     6121 acccacgcag gcgaggacgt ggcggtgttc gcgcgcggcc cgcaggcgca cctggttcac
     6181 ggcgtgcagg agcagacctt catagcgcac gtcatggcct tcgccgcctg cctggagccc
     6241 tacaccgcct gcgacctggc gccccccgcc ggcaccaccg acgccgcgca cccgggtggc
     6301 ggaggactga acgacatctt cgaggctcag aaaatcgaat ggcacgaaca tcaccatcac
     6361 catcaccatc accattaagg ccggcctgta gtcactggca caacgcgtta cccggtaagc
     6421 caatcgggta tacacggtcg tcatactgca gacagggttc ttctactttg caagatagtc
     6481 ttgagtagta aaataaatag atagagaaaa tcagcctcga ctgtgccttc tagttgccag
     6541 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     6601 gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc tcggtctatt
     6661 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6721 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     6781 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     6841 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     6901 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     6961 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     7021 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     7081 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     7141 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     7201 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     7261 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     7321 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     7381 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     7441 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     7501 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     7561 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     7621 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     7681 acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     7741 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacac
     7801 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     7861 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     7921 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     7981 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     8041 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     8101 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     8161 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     8221 cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa
     8281 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     8341 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     8401 cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8461 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8521 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8581 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     8641 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     8701 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8761 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8821 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     8881 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8941 acggaaatgt tgaatactca tattcttcct t
//
