LOCUS       sequence-478444-        8959 bp    DNA     circular SYN 24-JUL-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 8959)
  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 8959)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 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-SEMA7A-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8959
                     /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             4609..4632
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6292..6336
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6337..6363
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6503..6520)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6509..6620
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6738..6758)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6743..7072
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6923..7058
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6985..7004
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7120..7167
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7186..7785
                     /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(7186..7205)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7682..7702
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7915..8036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7952..7971)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8006..8025
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8089..8880,8881..8949))
                     /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     8712..8731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8950..8959,1..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 tcaaggacat
     2761 cttagaagcg gccctagaat cttcgccgtg tggaagggac acgtcggcca ggacagagtg
     2821 gatttcggac agaccgagcc tcacaccgtg ctgtttcacg agcctggctc tagctctgtg
     2881 tgggttggag gcagaggcaa ggtgtacctg ttcgacttcc ccgagggcaa gaatgcctct
     2941 gtgcggaccg tgaatatcgg cagcacaaag ggcagctgtc tggacaagag agactgcgag
     3001 aactacatca ccctgctgga aagaagaagc gagggcctgc tggcctgtgg caccaatgct
     3061 agacacccca gctgctggaa cctggtcaat ggcacagtgg tgcctctggg cgagatgaga
     3121 ggctacgccc cttttagccc cgacgagaat agcctggtgc tgttcgaagg cgacgaggtg
     3181 tacagcacca tccggaagca agagtacaac ggcaagatcc ccagattccg gcggatcaga
     3241 ggcgagagcg agctgtacac aagcgacacc gtgatgcaga accctcagtt catcaaggcc
     3301 accatcgtgc accaggacca ggcctacgac gacaagatct actacttctt cagagaggac
     3361 aaccccgaca agaaccccga ggctcctctg aacgtgtcaa gagtggccca gctgtgcaga
     3421 ggcgatcaag gcggagaaag cagcctgagc gtgtccaagt ggaacacctt cctgaaggcc
     3481 atgctcgtgt gttccgatgc cgccaccaac aagaacttca accggctgca ggatgtgttc
     3541 ctgctgcctg atccttctgg ccagtggcgg gatacaagag tgtacggcgt gttcagcaac
     3601 ccctggaact acagcgccgt gtgcgtgtac agcctgggcg acatcgacaa ggtgttcaga
     3661 acctccagcc tgaagggcta ccacagcagc ctgcctaatc ctcggcctgg caagtgtctg
     3721 ccagaccagc agcctattcc taccgagaca ttccaggtgg ccgacagaca tcctgaggtg
     3781 gcccaacgag tggaacctat gggccctctg aaaacccctc tgttccacag caagtaccac
     3841 taccagaaag tggccgtgca cagaatgcag gcctctcacg gcgagacttt ccacgtgctg
     3901 tacctgacca ccgacagggg cacaatccac aaggttgtgg aacccggcga gcaagagcac
     3961 agcttcgcct tcaacatcat ggaaatccag cctttcagac gggccgctgc catccagaca
     4021 atgtctctgg atgccgagcg gcggaagctg tacgttagct ctcagtggga agtgtcccag
     4081 gtgccactgg atctgtgcga agtgtatggc ggcggatgtc acggctgtct gatgagcaga
     4141 gatccctact gcggctggga tcagggcaga tgcatcagca tctacagcag cgagagaagc
     4201 gtgctgcaga gcatcaatcc tgccgagcca cacaaagagt gccccaatcc taagcctgac
     4261 aaggcccctc tgcagaaagt gtctctggcc cctaacagcc ggtactacct gagctgcccc
     4321 atggaaagca gacacgccac ctacagctgg cggcacaaag aaaacgtgga acagtcctgc
     4381 gagcccggcc accagtctcc aaattgcatc ctgttcatcg agaacctgac agcccagcag
     4441 tacggccact acttctgcga agcccaagag ggcagctact ttagagaggc ccagcactgg
     4501 cagctgctgc ccgaggatgg aattatggcc gagcatctgc tgggacacgc ctgtggcgcg
     4561 ccggaagatc aagttgatcc aagattgatt gatggcaagg gcggatctct ggaggtgctg
     4621 ttccagggac caggcggaag cggcaatccc ggcggcgatc tggcccccca gatgctgcgc
     4681 gagctgcagg agaccaatgc cgccctgcag gacgtgcgcg agctgctgcg ccaccgcgtg
     4741 aaggagatca ccttcctgaa gaacaccgtg atggagtgcg acgcctgcgg tatgcagccc
     4801 tctagaggct ccggctccat catcccagtt gaggaggaga acccggactt ctggaaccgc
     4861 gaggcagccg aggccctggg tgccgccaag aagctgcagc ctgcacagac agccgccaag
     4921 aacctcatca tcttcctggg cgatgggatg ggggtgtcta cggtgacagc tgccaggatc
     4981 ctaaaagggc agaagaagga caaactgggg cctgagatac ccctggccat ggaccgcttc
     5041 ccatatgtgg ctctgtccaa gacatacaat gtagacaaac atgtgccaga cagtggagcc
     5101 acagccacgg cctacctgtg cggggtcaag ggcaacttcc agaccattgg cttgagtgca
     5161 gccgcccgct ttaaccagtg caacacgaca cgcggcaacg aggtcatctc cgtgatgaat
     5221 cgggccaaga aagcagggaa gtcagtggga gtggtaacca ccacacgagt gcagcacgcc
     5281 tcgccagccg gcacctacgc ccacacggtg aaccgcaact ggtactcgga cgccgacgtg
     5341 cctgcctcgg cccgccagga ggggtgccag gacatcgcta cgcagctcat ctccaacatg
     5401 gacattgacg tgatcctagg tggaggccga aagtacatgt ttcgcatggg aaccccagac
     5461 cctgagtacc cagatgacta cagccaaggt gggaccaggc tggacgggaa gaatctggtg
     5521 caggaatggc tggcgaagcg ccagggtgcc cggtatgtgt ggaaccgcac tgagctcatg
     5581 caggcttccc tggacccgtc tgtgacccat ctcatgggtc tctttgagcc tggagacatg
     5641 aaatacgaga tccaccgaga ctccacactg gacccctccc tgatggagat gacagaggct
     5701 gccctgcgcc tgctgagcag gaacccccgc ggcttcttcc tcttcgtgga gggtggtcgc
     5761 atcgaccatg gtcatcatga aagcagggct taccgggcac tgactgagac gatcatgttc
     5821 gacgacgcca ttgagagggc gggccagctc accagcgagg aggacacgct gagcctcgtc
     5881 actgccgacc actcccacgt cttctccttc ggaggctacc ccctgcgagg gagctccatc
     5941 ttcgggctgg cccctggcaa ggcccgggac aggaaggcct acacggtcct cctatacgga
     6001 aacggtccag gctatgtgct caaggacggc gcccggccgg atgttaccga gagcgagagc
     6061 gggagccccg agtatcggca gcagtcagca gtgcccctgg acgaagagac ccacgcaggc
     6121 gaggacgtgg cggtgttcgc gcgcggcccg caggcgcacc tggttcacgg cgtgcaggag
     6181 cagaccttca tagcgcacgt catggccttc gccgcctgcc tggagcccta caccgcctgc
     6241 gacctggcgc cccccgccgg caccaccgac gccgcgcacc cgggtggcgg aggactgaac
     6301 gacatcttcg aggctcagaa aatcgaatgg cacgaacatc accatcacca tcaccatcac
     6361 cattaaggcc ggcctgtagt cactggcaca acgcgttacc cggtaagcca atcgggtata
     6421 cacggtcgtc atactgcaga cagggttctt ctactttgca agatagtctt gagtagtaaa
     6481 ataaatagat agagaaaatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6541 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6601 taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct tttgatttat
     6661 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6721 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     6781 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     6841 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     6901 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6961 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     7021 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     7081 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     7141 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatgac cgagtacaag
     7201 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     7261 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     7321 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     7381 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     7441 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     7501 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     7561 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     7621 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac atccgcgccc
     7681 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     7741 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat
     7801 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     7861 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     7921 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     7981 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     8041 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt
     8101 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     8161 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat
     8221 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg
     8281 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     8341 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     8401 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     8461 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8521 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8581 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8641 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8701 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8761 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8821 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8881 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8941 aatactcata ttcttcctt
//
