LOCUS       sequence-433193-        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-KIR2DS4-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     113..132
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     138..157
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      243..831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     732..751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     985..1002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1100..1117
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1101..1117
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1573..1589
                     /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        1756..2135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2136..2339
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2290..2310
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2336..2360
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2570..2588
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2570..2587
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2601..2620
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4109..4153
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4154..4171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4311..4328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4317..4428
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4546..4566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4551..4880
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4731..4866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4793..4812
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4928..4975
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4994..5593
                     /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(4994..5013)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5490..5510
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5723..5844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5760..5779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5814..5833
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5897..6688,6689..6757))
                     /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     6520..6539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6758..6778,1..71))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
       61 aaataaacaa ataggggtca gtgttacaac caattaacca attctgaaca ttatcgcgag
      121 cccatttata cctgaatatg gctcataaca ccccttgctc atgaccaaaa tcccttaacg
      181 tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
      241 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
      301 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      361 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt agcccaccac
      421 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      481 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      541 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      601 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      661 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      721 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      781 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      841 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      901 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      961 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aggcgagagt
     1021 agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct ttttgcgttt
     1081 ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt aagctcgggc cccctgggcg
     1141 gttctgataa cgagtaatcg ttaatccgca aataacgtaa aaacccgctt cggcgggttt
     1201 ttttatgggg ggagtttagg gaaagagcat ttgtcagaat atttaagggc gcctgtcact
     1261 ttgcttgata tatgagaatt atttaacctt ataaatgaga aaaaagcaac gcactttaaa
     1321 taagatacgt tgctttttcg attgatgaac acctataatt aaactattca tctattattt
     1381 atgatttttt gtatatacaa tatttctagt ttgttaaaga gaattaagaa aataaatctc
     1441 gaaaataata aagggaaaat cagtttttga tatcaaaatt atacatgtca acgataatac
     1501 aaaatataat acaaactata agatgttatc agtatttatt atcatttaga ataaattttg
     1561 tgtcgccctt aattgtgagc ggataacaat tacgagcttc atgcacagtg gcgttgactc
     1621 aatattggcc attagccata ttattcattg gttatatagc ataaatcaat attggctatt
     1681 ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc tcatgtccaa
     1741 tatgaccgcc atgttggcat tgattattga ctagttatta atagtaatca attacggggt
     1801 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1861 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1921 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1981 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2041 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2101 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2161 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2221 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2281 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     2341 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     2401 gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg cggattcccc
     2461 gtgccaagag tgacgtaagt accgcctata gagtctatag gcccaccccc ttggcttcgt
     2521 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     2581 actatagaat aacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact
     2641 gcacctcggt tctatcgaaa acgcgcctgc tagcgccacc atgaagacca tcattgccct
     2701 gagctacatc ttctgcctgg tgttcgctgc ggccgcccaa gaaggcgtgc acagaaagcc
     2761 atcttttctg gccctgcctg gccacctggt caagagcgaa gaaaccgtga tcctgcagtg
     2821 ttggagcgac gtgatgttcg agcacttcct gctgcacaga gagggcaagt tcaacaacac
     2881 cctgcacctg atcggagagc accacgatgg cgtgtccaag gccaatttca gcatcggccc
     2941 tatgatgcct gtgctggccg gcacatacag atgttacggc tctgtgcctc acagccccta
     3001 tcagctgtct gcccctagcg atcccctgga catggtcatc atcggcctgt acgagaagcc
     3061 ttctctgagt gcccagcctg gacctacagt gcaggctggc gaaaatgtga ccctgagctg
     3121 tagcagccgg tccagctacg atatgtacca cctgagtaga gagggcgaag cccacgaaag
     3181 acgactgcct gctgtgcgga gcatcaatgg caccttccag gccgattttc cactgggacc
     3241 tgccacacac ggcggcacct atagatgctt cggcagcttc agagatgccc cttacgagtg
     3301 gtccaacagc agcgatcctc tgctggtgtc tgtgaccggc aatcccagca atagctggcc
     3361 ttctccaacc gagcctagca gcaagacagg caaccccaga catctgcatg gcgcgccggg
     3421 cggcgacaag acccacacct gccccccctg cccagctcca gaactgctgg gagggccttc
     3481 cgtgttcctg ttccccccca agcccaagga caccctgatg atcagcagga cccccgaggt
     3541 gacctgcgtg gtggtggccg tgtcccacga ggacccagag gtgaagttca actggtacgt
     3601 ggacggcgtg gaggtgcaca acgccaagac caagcccaga gaggagcagt acaacagcac
     3661 ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagaata
     3721 caagtgcaaa gtctccaaca aggccctggc tgccccaatc gaaaagacaa tcagcaaggc
     3781 caagggccag ccacgggagc cccaggtgta caccctgccc cccagccggg aggagatgac
     3841 caagaaccag gtgtccctga cctgtctggt gaagggcttc taccccagcg atatcgccgt
     3901 ggagtgggag agcaacggcc agcccgagaa caactacaag accacccccc cagtgctgga
     3961 cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg gacaagtcca ggtggcagca
     4021 gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa
     4081 gtccctgagc ctgagccccg gcggcagcgg tcttaatgat attttcgagg ctcagaagat
     4141 tgagtggcat gaacaccacc accatcacca ctaaggccgg cctgtagtca ctggcacaac
     4201 gcgttacccg gtaagccaat cgggtataca cggtcgtcat actgcagaca gggttcttct
     4261 actttgcaag atagtcttga gtagtaaaat aaatagatag agaaaatcag cctcgactgt
     4321 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     4381 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac
     4441 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4501 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4561 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4621 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4681 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4741 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4801 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4861 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     4921 agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     4981 gaggaactaa accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     5041 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     5101 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     5161 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     5221 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     5281 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     5341 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     5401 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     5461 gcccgccttc ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     5521 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     5581 gcccggtgcc tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt
     5641 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5701 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     5761 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5821 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     5881 ggcgtaatca tggtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5941 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6001 gcttaccatc tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg
     6061 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6121 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6181 ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt
     6241 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6301 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6361 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6421 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6481 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6541 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6601 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6661 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6721 agggaataag ggcgacacgg aaatgttgaa tactcatatt cttccttttt caatatta
//
