LOCUS       sequence-303139-        9037 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 9037)
  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 9037)
  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-CNTN6-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..9037
                     /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             complement(4789..4803)
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             6379..6423
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6424..6441
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6581..6598)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6587..6698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6816..6836)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6821..7150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7001..7136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7063..7082
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7198..7245
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7264..7863
                     /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(7264..7283)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7760..7780
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7993..8114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8030..8049)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8084..8103
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8167..8958,8959..9027))
                     /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     8790..8809
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9028..9037,1..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 cggccgccga tggactgctg
     2761 agcagaccta tctttaccca agagcctcac gacgtgatct tcccactgga cctgagcaag
     2821 agcgaagtga tcctgaattg cgccgccaac ggctacccat ctccacacta cagatggaag
     2881 cagaacggca ccgacatcga cttcaccatg agctaccact acaggctgga tggcggcagc
     2941 ctggccatca atagccctca cacagaccag gacatcggca tgtaccagtg tctggccacc
     3001 aatctgctgg gcaccattct gagcagaaag gccaagctgc agttcgccta catcgaggac
     3061 ttcgagacaa agaccagatc caccgtgtcc gttagagaag gccaaggcgt tgtgctgctg
     3121 tgtggccctc ctcctcattt cggcgatctg agctacgcct ggaccttcaa cgacaaccct
     3181 ctgtacgtgc aagaggacaa ccgcagattc gtgtcccaag agacaggcaa cctgtatatc
     3241 gccaaggtgg aacccagcga cgtgggcaac tacacctgtt tcatcaccaa caaagaggcc
     3301 cagcggagcg ttcagggacc tcctacacct ctggttcaga gaacagatgg cgtgatgggc
     3361 gagtacgagc ccaagatcga agtgcggttc cctgagacaa tccaggccgc caaggatagc
     3421 agcgtgaagc tggaatgctt cgccctgggc aaccctgtgc ctgacatttc ttggagaagg
     3481 ctggacggaa gccctctgcc tggcaaagtg aagtacagca agagccaggc catcctggaa
     3541 atccccaact tccagcaaga ggatgagggc ttctacgagt gtatcgccag caacctgaga
     3601 ggcagaaatc tggccaaggg ccagctgatc ttttacgccc ctcctgagtg ggagcagaag
     3661 atccagaata cccacctgag catctacgac aacctgctgt gggagtgcaa ggccagcgga
     3721 aagcctaatc cttggtacac ctggctgaag aacggcgaga gactgaaccc cgaggaaaga
     3781 atccagatcg agaatggcac cctgatcatc accatgctga acgtgtccga cagcggagtg
     3841 tatcagtgtg ccgccgagaa caagtaccag atcatctacg ccaacgccga gctgagagtg
     3901 ctggcctccg ctcctgattt cagcaagtcc cctgtgaaga aaaagtcctt cgtgcaagtc
     3961 ggcggcgaca tcgtgatcgg ctgcaagcca aatgcctttc ctagagccgc catcagctgg
     4021 aagagaggca cagagacact gcggcagagc aagagaatct ttctgctgga agatggcagc
     4081 ctgaagatct acaacatcac cagaagcgac gccggcagct acacatgcat tgccacaaat
     4141 cagttcggca ccgccaagaa caccggaagc ctgatcgtga aagaacggac cgtgatcacc
     4201 gtgcctccaa gcaagatgga tgtgaccgtg ggcgagagca tcgtgctgcc ttgtcaagtg
     4261 tcccacgatc ctagcatcga ggtggtgttc gtgtggttct tcaacgggga cgtgatcgac
     4321 ctgaagaaag gcgtggccca ctttgagaga atcggcggag aatctgtggg cgacctgatg
     4381 atccggaaca tccagctgca ccacagcggc aagtacctgt gtaccgtgca gaccacactg
     4441 gaatctctga gcgccgtggc cgatatcatc gttagaggac ctccaggacc acctgaggac
     4501 gtgcaggtcg aggatatcag cagcaccaca agccagctgt cttggagagc cggacctgac
     4561 aacaacagcc ccatccagat cttcacaatc cagaccagaa cacccttcag cgttggatgg
     4621 caggctgtgg ccactgtgcc agagattctg aacggcaaga cctacaacgc caccgtcgtt
     4681 ggactgagcc cttgggtcga gtatgagttt agagtggtgg ccggcaacag catcggaatc
     4741 ggagagcctt ctgagcctag cgagctgctg agaacaaaag cctctgtgcc agtggtggct
     4801 cccgtgaata tccatggtgg tggcggaagc agatccgagc tggtcatcac atgggagagc
     4861 atccctgagg aactgcagaa tggcgaaggc ttcggctaca tcatcatgtt cagacccgtg
     4921 ggctccacca cctggtccaa agaaaaggtg tccagcgtgg aaagcagccg cttcgtgtac
     4981 cggaatgaga gcatcatccc tctgagcccc ttcgaagtga aagtgggcgt gtacaacaac
     5041 gaaggcgagg gcagcctgtc taccgtgaca atcgtgtact ctggcgagga tgagccccag
     5101 ctggctccta gaggaacaag cctgcagagc tttagcgcca gcgagatgga agtgtcctgg
     5161 aacgccattg cctggaacag aaacacaggc cgcgtgctgg gctacgaggt gctgtactgg
     5221 accgacgaca gcaaagaatc tatgatcggc aagatcagag tgtccggcaa cgtgaccacc
     5281 aagaacatca ccggcctgaa ggccaacacc atctactttg cctccgtgcg ggcctacaat
     5341 accgccggaa caggacctag cagccctcct gtgaatgtga ccacaaaaaa gagccctcca
     5401 agccagcctc ctgccaatat cgcctggaag ctgaccaaca gcaagctgtg tctgaactgg
     5461 gagcacgtca agaccatgga aaacgagagc gaggtcctgg gctacaagat cctgtaccgg
     5521 cagaaccggc agtctaagac ccacattctg gaaacaaaca acaccagcgc cgaactgctg
     5581 gtgcccttcg aggaagatta cctgatcgag atccggaccg tgtctgacgg cggagatgga
     5641 tctagcagcg aggaaatcag aatccccaag atgtcctctg gcgcgccggg cggcgacaag
     5701 acccacacct gccccccctg cccagctcca gaactgctgg gagggccttc cgtgttcctg
     5761 ttccccccca agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg
     5821 gtggtggccg tgtcccacga ggacccagag gtgaagttca actggtacgt ggacggcgtg
     5881 gaggtgcaca acgccaagac caagcccaga gaggagcagt acaacagcac ctacagggtg
     5941 gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagaata caagtgcaaa
     6001 gtctccaaca aggccctggc tgccccaatc gaaaagacaa tcagcaaggc caagggccag
     6061 ccacgggagc cccaggtgta caccctgccc cccagccggg aggagatgac caagaaccag
     6121 gtgtccctga cctgtctggt gaagggcttc taccccagcg atatcgccgt ggagtgggag
     6181 agcaacggcc agcccgagaa caactacaag accacccccc cagtgctgga cagcgacggc
     6241 agcttcttcc tgtacagcaa gctgaccgtg gacaagtcca ggtggcagca gggcaacgtg
     6301 ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc
     6361 ctgagccccg gcggcagcgg tcttaatgat attttcgagg ctcagaagat tgagtggcat
     6421 gaacaccacc accatcacca ctaaggccgg cctgtagtca ctggcacaac gcgttacccg
     6481 gtaagccaat cgggtataca cggtcgtcat actgcagaca gggttcttct actttgcaag
     6541 atagtcttga gtagtaaaat aaatagatag agaaaatcag cctcgactgt gccttctagt
     6601 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     6661 cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac cctatctcgg
     6721 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     6781 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     6841 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     6901 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     6961 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     7021 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     7081 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     7141 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
     7201 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     7261 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     7321 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     7381 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     7441 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     7501 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     7561 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     7621 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     7681 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     7741 ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     7801 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     7861 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     7921 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     7981 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     8041 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     8101 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     8161 tggtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     8221 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     8281 tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg atttatcagc
     8341 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     8401 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     8461 gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     8521 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     8581 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     8641 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     8701 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     8761 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     8821 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     8881 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     8941 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     9001 ggcgacacgg aaatgttgaa tactcatatt cttcctt
//
