LOCUS       sequence-304093-        8989 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 8989)
  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 8989)
  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-LRRN2-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8989
                     /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             4594..4629
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4639..4662
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6322..6366
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6367..6393
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6533..6550)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6539..6650
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6768..6788)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6773..7102
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6953..7088
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7015..7034
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7150..7197
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7216..7815
                     /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(7216..7235)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7712..7732
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7945..8066
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7982..8001)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8036..8055
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8119..8910,8911..8979))
                     /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     8742..8761
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8980..8989,1..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 cggccgccgc tgttcctgtt
     2761 gttccttggc atgtgccctg tcctccacag tgcgcctgtc agatcagacc ctggtacacc
     2821 cctcggagca gctacagaga agccaccacc gtggactgca acgacctgtt tctgacagcc
     2881 gtgcctcctg ctctgcctgc cggaactcaa acactgctgc tgcagagcaa cagcatcgtg
     2941 cgcgtggacc agtctgagct gggctatctg gccaatctga ccgagctgga cctgagccag
     3001 aacagcttta gcgacgcccg ggactgcgat tttcatgccc tgcctcaact gctgagcctg
     3061 cacctggaag agaaccagct gacccggctg gaagatcact cttttgccgg actggccagc
     3121 ctgcaagagc tgtacctgaa tcacaaccag ctgtaccgga tcgcccctag agcctttagc
     3181 ggcctgagca acctgctgcg gctgcatctg aacagcaatc tgctgagagc catcgacagc
     3241 cgttggttcg agatgctgcc caacctggaa atcctgatga tcggcggcaa caaggtggac
     3301 gccatcctgg acatgaactt cagacccctg gccaacctga gaagcctggt gctggctggc
     3361 atgaacctga gagagatcag cgactacgcc ctggaaggcc tgcagagcct ggaaagcctg
     3421 tccttctacg acaatcagct ggccagagtg cctcggagag cactggaaca agtgcccggc
     3481 ctgaagttcc tggatctgaa caagaatccc ctgcagagag tcggccctgg cgacttcgct
     3541 aatatgctgc acctcaaaga gctgggcctg aacaacatgg aagaactggt gtccatcgac
     3601 aagttcgccc tggtcaatct gcccgaactg acaaagctgg acatcaccaa caatccccgg
     3661 ctgagcttca ttcaccccag agccttccac catctgcctc agatggaaac tctgatgctc
     3721 aacaacaacg ccctgagcgc cctgcaccag cagacagttg agagcctgcc taacctgcaa
     3781 gaagtgggcc tgcacggcaa ccccatcaga tgcgattgcg tgatcagatg ggccaacgcc
     3841 acaggcacca gagtgcggtt tatcgagcct cagagcaccc tgtgtgccga gcctccagat
     3901 ctgcagagac tgccagtcag agaggtgccc ttcagagaga tgaccgacca ctgcctgcct
     3961 ctgatcagcc ccagatcttt cccacctagc ctgcaggttg ccagcggcga atctatggtg
     4021 ctgcactgta gagccctggc cgagcctgag cctgagatct attgggttac accagccggc
     4081 ctgagactga caccagctca tgctggcaga cggtacagag tgtacccaga gggcacactg
     4141 gaactgagaa gagtgacagc cgaggaagcc ggactgtaca cttgcgtggc ccagaatctt
     4201 gtgggcgccg ataccaagac cgtgtccgtg gttgttggca gagcactgct ccagcctggc
     4261 agagatgaag gccagggact cgagctgaga gtgcaagaga cacaccccta ccacatcctg
     4321 ctgtcctggg tcacccctcc taataccgtg tccaccaacc tgacctggtc tagcgccagc
     4381 tctctgagag gacaaggcgc tacagctctg gctagactgc ctagaggcac ccacagctac
     4441 aacatcacca gactgctgca ggccaccgag tactgggctt gtctgcaagt ggccttcgcc
     4501 gatgctcata cccagctggc ttgtgtgtgg gccagaacaa aagaggccac cagctgtcat
     4561 agagccctcg gcgatagacc cggtggcgcg ccggaagatc aagttgatcc aagattgatt
     4621 gatggcaagg gcggatctct ggaggtgctg ttccagggac caggcggaag cggcaatccc
     4681 ggcggcgatc tggcccccca gatgctgcgc gagctgcagg agaccaatgc cgccctgcag
     4741 gacgtgcgcg agctgctgcg ccaccgcgtg aaggagatca ccttcctgaa gaacaccgtg
     4801 atggagtgcg acgcctgcgg tatgcagccc tctagaggct ccggctccat catcccagtt
     4861 gaggaggaga acccggactt ctggaaccgc gaggcagccg aggccctggg tgccgccaag
     4921 aagctgcagc ctgcacagac agccgccaag aacctcatca tcttcctggg cgatgggatg
     4981 ggggtgtcta cggtgacagc tgccaggatc ctaaaagggc agaagaagga caaactgggg
     5041 cctgagatac ccctggccat ggaccgcttc ccatatgtgg ctctgtccaa gacatacaat
     5101 gtagacaaac atgtgccaga cagtggagcc acagccacgg cctacctgtg cggggtcaag
     5161 ggcaacttcc agaccattgg cttgagtgca gccgcccgct ttaaccagtg caacacgaca
     5221 cgcggcaacg aggtcatctc cgtgatgaat cgggccaaga aagcagggaa gtcagtggga
     5281 gtggtaacca ccacacgagt gcagcacgcc tcgccagccg gcacctacgc ccacacggtg
     5341 aaccgcaact ggtactcgga cgccgacgtg cctgcctcgg cccgccagga ggggtgccag
     5401 gacatcgcta cgcagctcat ctccaacatg gacattgacg tgatcctagg tggaggccga
     5461 aagtacatgt ttcgcatggg aaccccagac cctgagtacc cagatgacta cagccaaggt
     5521 gggaccaggc tggacgggaa gaatctggtg caggaatggc tggcgaagcg ccagggtgcc
     5581 cggtatgtgt ggaaccgcac tgagctcatg caggcttccc tggacccgtc tgtgacccat
     5641 ctcatgggtc tctttgagcc tggagacatg aaatacgaga tccaccgaga ctccacactg
     5701 gacccctccc tgatggagat gacagaggct gccctgcgcc tgctgagcag gaacccccgc
     5761 ggcttcttcc tcttcgtgga gggtggtcgc atcgaccatg gtcatcatga aagcagggct
     5821 taccgggcac tgactgagac gatcatgttc gacgacgcca ttgagagggc gggccagctc
     5881 accagcgagg aggacacgct gagcctcgtc actgccgacc actcccacgt cttctccttc
     5941 ggaggctacc ccctgcgagg gagctccatc ttcgggctgg cccctggcaa ggcccgggac
     6001 aggaaggcct acacggtcct cctatacgga aacggtccag gctatgtgct caaggacggc
     6061 gcccggccgg atgttaccga gagcgagagc gggagccccg agtatcggca gcagtcagca
     6121 gtgcccctgg acgaagagac ccacgcaggc gaggacgtgg cggtgttcgc gcgcggcccg
     6181 caggcgcacc tggttcacgg cgtgcaggag cagaccttca tagcgcacgt catggccttc
     6241 gccgcctgcc tggagcccta caccgcctgc gacctggcgc cccccgccgg caccaccgac
     6301 gccgcgcacc cgggtggcgg aggactgaac gacatcttcg aggctcagaa aatcgaatgg
     6361 cacgaacatc accatcacca tcaccatcac cattaaggcc ggcctgtagt cactggcaca
     6421 acgcgttacc cggtaagcca atcgggtata cacggtcgtc atactgcaga cagggttctt
     6481 ctactttgca agatagtctt gagtagtaaa ataaatagat agagaaaatc agcctcgact
     6541 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     6601 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc acaacactca
     6661 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6721 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     6781 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     6841 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     6901 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     6961 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     7021 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     7081 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     7141 tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag
     7201 gtgaggaact aaaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     7261 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     7321 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     7381 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     7441 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     7501 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     7561 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     7621 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     7681 gtgcccgcct tcctggagac atccgcgccc cgcaacctcc ccttctacga gcggctcggc
     7741 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     7801 aagcccggtg cctgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
     7861 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     7921 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     7981 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     8041 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     8101 ttggcgtaat catggtcatt accaatgctt aatcagtgag gcacctatct cagcgatctg
     8161 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     8221 gggcttacca tctggcccca gcgctgcgat gataccgcga gaaccacgct caccggctcc
     8281 ggatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     8341 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     8401 agttaatagt ttgcgcaacg ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc
     8461 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     8521 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8581 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8641 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8701 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8761 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8821 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8881 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8941 aaagggaata agggcgacac ggaaatgttg aatactcata ttcttcctt
//
