LOCUS       sequence-433195-        6778 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 6778)
  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 6778)
  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-KIR2DS5-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     104..123
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     129..148
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      234..822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     723..742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     976..993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1091..1108
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1092..1108
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1564..1580
                     /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        1747..2126
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2127..2330
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2281..2301
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2327..2351
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2561..2579
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2561..2578
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2592..2611
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4100..4144
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4145..4162
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4302..4319)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4308..4419
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4537..4557)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4542..4871
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4722..4857
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4784..4803
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4919..4966
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4985..5584
                     /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(4985..5004)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5481..5501
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5714..5835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5751..5770)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5805..5824
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5888..6679,6680..6748))
                     /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     6511..6530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6749..6778,1..62))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
       61 aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga gcccatttat
      121 acctgaatat ggctcataac accccttgct catgaccaaa atcccttaac gtgagttacg
      181 cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      241 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      301 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      361 gcgcagatac caaatactgt tcttctagtg tagccgtagt tagcccacca cttcaagaac
      421 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      481 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      541 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      601 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      661 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      721 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      781 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      841 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      901 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      961 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaggcgagag tagggaactg
     1021 ccaggcatca aactaagcag aaggcccctg acggatggcc tttttgcgtt tctacaaact
     1081 ctttctgtgt tgtaaaacga cggccagtct taagctcggg ccccctgggc ggttctgata
     1141 acgagtaatc gttaatccgc aaataacgta aaaacccgct tcggcgggtt tttttatggg
     1201 gggagtttag ggaaagagca tttgtcagaa tatttaaggg cgcctgtcac tttgcttgat
     1261 atatgagaat tatttaacct tataaatgag aaaaaagcaa cgcactttaa ataagatacg
     1321 ttgctttttc gattgatgaa cacctataat taaactattc atctattatt tatgattttt
     1381 tgtatataca atatttctag tttgttaaag agaattaaga aaataaatct cgaaaataat
     1441 aaagggaaaa tcagtttttg atatcaaaat tatacatgtc aacgataata caaaatataa
     1501 tacaaactat aagatgttat cagtatttat tatcatttag aataaatttt gtgtcgccct
     1561 taattgtgag cggataacaa ttacgagctt catgcacagt ggcgttgact caatattggc
     1621 cattagccat attattcatt ggttatatag cataaatcaa tattggctat tggccattgc
     1681 atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca atatgaccgc
     1741 catgttggca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1801 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1861 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1921 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1981 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     2041 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     2101 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     2161 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     2221 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     2281 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
     2341 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
     2401 accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga
     2461 gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttcg ttagaacgcg
     2521 gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa
     2581 taacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcacctcgg
     2641 ttctatcgaa aacgcgcctg ctagcgccac catgaagacc atcattgccc tgagctacat
     2701 cttctgcctg gtgttcgctg cggccgccca tgagggcttt agaagaaagc cttctctgct
     2761 ggctcacccc ggacctctgg tcaagtctga agagacagtg atcctgcagt gttggagcga
     2821 cgtgatgttc gagcacttcc tgctgcacag agagggcacc ttcaatcaca ccctgagact
     2881 gatcggcgag cacatcgatg gcgtgtccaa gggcaatttc agcatcggca gaatgaccca
     2941 ggatctggcc ggcacctaca gatgttacgg ctctgtgaca cacagcccct accagctgtc
     3001 tgcccctagc gatcctctgg atatcgtgat caccggcctg tacgagaagc ctagtctgcc
     3061 tgctcagcct ggacctacag ttctggctgg cgaaagcgtg accctgagct gtagcagcag
     3121 atccagctac gacatgtacc acctgagtag agagggcgaa gcccacgaaa gaaggctgcc
     3181 tgccggacct aaagtgaaca gaaccttcca ggccgactct cctctggacc ctgctacaca
     3241 tggcggcgct tacagatgct tcggcagctt cagagacagc ccttacgagt ggtccaagag
     3301 cagcgatcca ctgctggtgt ctgtgaccgg caacagcagc aatagctggc cttctccaac
     3361 cgagcctagc agcgagacag gcaaccccag acatctgcat ggcgcgccgg gcggcgacaa
     3421 gacccacacc tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct
     3481 gttccccccc aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt
     3541 ggtggtggcc gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt
     3601 ggaggtgcac aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt
     3661 ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa
     3721 agtctccaac aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca
     3781 gccacgggag ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca
     3841 ggtgtccctg acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga
     3901 gagcaacggc cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg
     3961 cagcttcttc ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt
     4021 gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag
     4081 cctgagcccc ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca
     4141 tgaacaccac caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc
     4201 ggtaagccaa tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa
     4261 gatagtcttg agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag
     4321 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4381 tcccactgtc ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg
     4441 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4501 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     4561 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4621 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4681 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4741 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4801 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4861 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt
     4921 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     4981 aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     5041 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     5101 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     5161 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     5221 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     5281 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     5341 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     5401 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     5461 cctggagaca tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     5521 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     5581 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5641 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5701 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     5761 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5821 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5881 atggtcatta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5941 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6001 ctggccccag cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag
     6061 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6121 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6181 tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6241 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6301 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6361 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6421 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6481 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6541 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6601 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6661 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6721 gggcgacacg gaaatgttga atactcatat tcttcctttt tcaatattat tgaagcat
//
