LOCUS       sequence-368526-        6577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6577)
  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 6577)
  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-VSTM4-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..37)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     264..284
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    402..513
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     502..519
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(659..676)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(677..721)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2009..2028)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2041..2059)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2042..2059)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2269..2293)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2290..2493)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2319..2339)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2494..2873
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    3040..3056
                     /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)."
     primer_bind     complement(3512..3529)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3512..3528)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3627..3644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3798..4386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3878..3897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4472..4491)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4497..4516)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4558..4649
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4650..4718,4719..5510)
                     /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     complement(4868..4887)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    5563..5684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5574..5593)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5628..5647
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5814..6413)
                     /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(5897..5917)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6394..6413
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6432..6479)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(join(6527..6577,1..279))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6541..6577,1..99)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ctcagaggca gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg
       61 cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac
      121 tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      181 ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
      241 tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagaa ttaattcgcg
      301 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
      361 tataaatcaa aagaatagac cgagataggg ttgagtgttg tgatgcaatt tcctcatttt
      421 attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc
      481 aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgattttctc tatctattta
      541 ttttactact caagactatc ttgcaaagta gaagaaccct gtctgcagta tgacgaccgt
      601 gtatacccga ttggcttacc gggtaacgcg ttgtgccagt gactacaggc cggccttagt
      661 ggtgatggtg gtggtgttca tgccactcaa tcttctgagc ctcgaaaata tcattaagac
      721 cgctgccgcc ggggctcagg ctcagggact tctgggtgta gtggttgtgc agggcctcgt
      781 gcatcacgct gcagctgaac acgttgccct gctgccacct ggacttgtcc acggtcagct
      841 tgctgtacag gaagaagctg ccgtcgctgt ccagcactgg gggggtggtc ttgtagttgt
      901 tctcgggctg gccgttgctc tcccactcca cggcgatatc gctggggtag aagcccttca
      961 ccagacaggt cagggacacc tggttcttgg tcatctcctc ccggctgggg ggcagggtgt
     1021 acacctgggg ctcccgtggc tggcccttgg ccttgctgat tgtcttttcg attggggcag
     1081 ccagggcctt gttggagact ttgcacttgt attctttgcc gttcagccag tcctggtgca
     1141 gcacggtcag cacggacacc accctgtagg tgctgttgta ctgctcctct ctgggcttgg
     1201 tcttggcgtt gtgcacctcc acgccgtcca cgtaccagtt gaacttcacc tctgggtcct
     1261 cgtgggacac ggccaccacc acgcaggtca cctcgggggt cctgctgatc atcagggtgt
     1321 ccttgggctt gggggggaac aggaacacgg aaggccctcc cagcagttct ggagctgggc
     1381 agggggggca ggtgtgggtc ttgtcgccgc ccggcgcgcc atacacatac aggtcctcga
     1441 agaaggccca ggtttctttg gtcttttcga agctgctttc ctcgctggcc ttcaggctga
     1501 tcactctcat ctctgtggcg ctgctgccat tagaccaggc ggtccacttg tttctgtgcc
     1561 ggctgatctc ttgcactctg cacacgtagt ggccctgatc gctaggctgc agtgtcagaa
     1621 cgctcagtct gtacagggcg cctctctgtt cttccagcag tctcagcctt ctccgcttgg
     1681 cgcttctgct gaagttgccg tagtactgca ccactctcag cttggtcatc ttgaccatca
     1741 gggcctcttg gctatcaaag ctgtgggcga agaaccaccg cacggccagc agagaatcct
     1801 ttcttctctt ctgggacacg tggcacagca gtgtggcatt ttcgccttcc aggtagtcca
     1861 ccacaggtcc aggagacact gtcacattca aggcggccgc agcgaacacc aggcagaaga
     1921 tgtagctcag ggcaatgatg gtcttcatgg tggcgctagc aggcgcgttt tcgatagaac
     1981 cgaggtgcag ttggacctgg gagtggacac ctgtggagag aaaggcaaag tggatgttat
     2041 tctatagtgt cacctaaatc gtatgtgtat gatacataag gttatgtatt aattgtagcc
     2101 gcgttctaac gaagccaagg gggtgggcct atagactcta taggcggtac ttacgtcact
     2161 cttggcacgg ggaatccgcg ttccaatgca ccgttcccgg ccgcggaggc tggatcggtc
     2221 ccggtgtctt ctatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt
     2281 cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     2341 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     2401 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     2461 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     2521 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     2581 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     2641 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     2701 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     2761 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     2821 aactaatgac cccgtaattg attactatta ataactagtc aataatcaat gccaacatgg
     2881 cggtcatatt ggacatgagc caatataaat gtacatatta tgatatagat acaacgtatg
     2941 caatggccaa tagccaatat tgatttatgc tatataacca atgaataata tggctaatgg
     3001 ccaatattga gtcaacgcca ctgtgcatga agctcgtaat tgttatccgc tcacaattaa
     3061 gggcgacaca aaatttattc taaatgataa taaatactga taacatctta tagtttgtat
     3121 tatattttgt attatcgttg acatgtataa ttttgatatc aaaaactgat tttcccttta
     3181 ttattttcga gatttatttt cttaattctc tttaacaaac tagaaatatt gtatatacaa
     3241 aaaatcataa ataatagatg aatagtttaa ttataggtgt tcatcaatcg aaaaagcaac
     3301 gtatcttatt taaagtgcgt tgcttttttc tcatttataa ggttaaataa ttctcatata
     3361 tcaagcaaag tgacaggcgc ccttaaatat tctgacaaat gctctttccc taaactcccc
     3421 ccataaaaaa acccgccgaa gcgggttttt acgttatttg cggattaacg attactcgtt
     3481 atcagaaccg cccagggggc ccgagcttaa gactggccgt cgttttacaa cacagaaaga
     3541 gtttgtagaa acgcaaaaag gccatccgtc aggggccttc tgcttagttt gatgcctggc
     3601 agttccctac tctcgccttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4201 ttcttgaagt ggtgggctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4381 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgacgcgcgc
     4441 gtaactcacg ttaagggatt ttggtcatga gcaaggggtg ttatgagcca tattcaggta
     4501 taaatgggct cgcgataatg ttcagaattg gttaattggt tgtaacactg acccctattt
     4561 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4621 tgcttcaata atattgaaaa aggaagaata tgagtattca acatttccgt gtcgccctta
     4681 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4741 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4801 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4861 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4921 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4981 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5041 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5101 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5161 taccaaacga cgagcgtgac accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac
     5221 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5281 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5341 ataaatccgg agccggtgag cgtggttctc gcggtatcat cgcagcgctg gggccagatg
     5401 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5461 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa tgaccatgat
     5521 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
     5581 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5641 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
     5701 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
     5761 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtagcac gtgtcaggca
     5821 ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg
     5881 acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggatgt ctccaggaag
     5941 gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc gctgcccaga
     6001 cccttgccct ggtggtcggg cgagacgccg acggtggcca ggaaccacgc gggctccttg
     6061 ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg ggaaccgctc
     6121 aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac gctctccggc
     6181 gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc acaccttgcc gatgtcgagc
     6241 ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg gcggtccgga
     6301 tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt gcgtacggcc
     6361 ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt catggtttag
     6421 ttcctcacct tgtcgtatta tactatgccg atatactatg ccgatgatta attgtcaaca
     6481 cgtgctgatc agatccgaaa atggatatac aagctcccgg gagctttttg caaaagccta
     6541 ggcctccaaa aaagcctcct cactacttct ggaatag
//
