LOCUS       sequence-302863-        7555 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 7555)
  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 7555)
  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-LRRC4C-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7555
                     /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             2835..2852
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             4897..4941
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4942..4959
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5099..5116)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5105..5216
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5334..5354)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5339..5668
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5519..5654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5581..5600
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5716..5763
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5782..6381
                     /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(5782..5801)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6278..6298
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6511..6632
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6548..6567)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6602..6621
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6685..7476,7477..7545))
                     /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     7308..7327
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7546..7555,1..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 cggccgccca aacatgtcct
     2761 agcgtgtgca gctgtagcaa ccagttcagc aaagtgatct gcgtgcggaa gaacctgaga
     2821 gaggtgccag acggcatcag caccaacacc agactgctga acctgcacga gaaccagatc
     2881 cagatcatca aagtgaacag cttcaagcac ctgaggcacc tggaaatcct gcagctgagc
     2941 cggaaccaca tcagaacaat cgagatcggc gccttcaacg gcctggccaa cctgaatacc
     3001 ctggaactgt tcgacaaccg gctgaccacc attcctaatg gcgccttcgt gtacctgagc
     3061 aagctgaaag agctgtggct gcggaacaac cccatcgagt ctatccccag ctacgccttc
     3121 aatagaatcc ccagcctgag aaggctggac ctgggagagc tgaagagact gagctatatc
     3181 agcgagggcg cctttgaggg cctgagcaac ctcagatacc tgaacctggc catgtgcaat
     3241 ctgagagaga tccccaatct gacccctctg atcaagctgg acgagctgga tctgagcggc
     3301 aaccacctgt ctgccatcag acctggaagt ttccagggcc tgatgcatct gcagaaactg
     3361 tggatgatcc agagccagat ccaagtgatc gagcggaacg ccttcgacaa tctgcagagc
     3421 ctggtggaaa tcaatctggc ccacaacaac ctgacactgc tgccccacga tctgttcaca
     3481 cctctgcacc acctggaacg catccatctg catcacaacc cctggaattg caactgcgac
     3541 atcctgtggc tgagctggtg gatcaaggac atggccccta gcaataccgc ctgctgcgcc
     3601 agatgcaaca cccctcctaa cctgaagggc agatatatcg gcgagctgga ccagaactac
     3661 ttcacctgtt acgcccctgt gatcgtggaa cctcctgccg acctgaatgt gacagaaggc
     3721 atggccgccg agctgaagtg tagagcctct accagcctga ccagcgtgtc ctggatcacc
     3781 cctaatggca cagtgatgac ccacggcgcc tacaaagtgc ggattgccgt gctgtccgac
     3841 ggcaccctga atttcacaaa cgtgaccgtg caggacaccg gcatgtacac ctgtatggtg
     3901 tccaacagcg tgggcaatac cacagccagc gccacactga acgtgacagc cgccacaacc
     3961 acacctttca gctacttcag caccgtgaca gtggaaacca tggaacccag ccaggatgag
     4021 gccagaacca ccgataacaa cgtgggccct acacctgtgg tggactggga aaccaccaac
     4081 gtgaccacaa gcctgacacc tcagagcacc agaagcaccg agaaaacctt cacaatcccc
     4141 gtgaccgaca tcaacagcgg catccctggc atcgacgaag tgatgaagac caccaaaggc
     4201 gcgccgggcg gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga
     4261 gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc
     4321 cccgaggtga cctgcgtggt ggtggccgtg tcccacgagg acccagaggt gaagttcaac
     4381 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac
     4441 aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc
     4501 aaagaataca agtgcaaagt ctccaacaag gccctggctg ccccaatcga aaagacaatc
     4561 agcaaggcca agggccagcc acgggagccc caggtgtaca ccctgccccc cagccgggag
     4621 gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat
     4681 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca
     4741 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagtccagg
     4801 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
     4861 acccagaagt ccctgagcct gagccccggc ggcagcggtc ttaatgatat tttcgaggct
     4921 cagaagattg agtggcatga acaccaccac catcaccact aaggccggcc tgtagtcact
     4981 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg
     5041 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc
     5101 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     5161 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa
     5221 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5281 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     5341 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5401 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     5461 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5521 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5581 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     5641 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     5701 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     5761 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     5821 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     5881 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     5941 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     6001 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     6061 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     6121 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     6181 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     6241 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg
     6301 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     6361 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat
     6421 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     6481 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     6541 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     6601 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     6661 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc
     6721 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6781 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     6841 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6901 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6961 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     7021 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7081 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7141 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7201 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7261 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7321 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7381 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7441 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7501 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt
//
