LOCUS       sequence-433180-        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-KIR2DL1-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     148..167
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      253..841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     742..761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     995..1012
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1110..1127
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1111..1127
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1583..1599
                     /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        1766..2145
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2146..2349
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2300..2320
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2346..2370
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2580..2598
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2580..2597
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2611..2630
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4119..4163
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4164..4181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4321..4338)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4327..4438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4556..4576)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4561..4890
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4741..4876
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4803..4822
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4938..4985
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5004..5603
                     /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(5004..5023)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5500..5520
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5733..5854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5770..5789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5824..5843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5907..6698,6699..6767))
                     /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     6530..6549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6768..6778,1..81))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
       61 gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca attctgaaca
      121 ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgctc atgaccaaaa
      181 tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
      241 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      301 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      361 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      421 agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      481 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      541 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      601 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      661 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      721 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      781 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      841 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      901 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
      961 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1021 aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct
     1081 ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt aagctcgggc
     1141 cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa aaacccgctt
     1201 cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat atttaagggc
     1261 gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga aaaaagcaac
     1321 gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt aaactattca
     1381 tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga gaattaagaa
     1441 aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt atacatgtca
     1501 acgataatac aaaatataat acaaactata agatgttatc agtatttatt atcatttaga
     1561 ataaattttg tgtcgccctt aattgtgagc ggataacaat tacgagcttc atgcacagtg
     1621 gcgttgactc aatattggcc attagccata ttattcattg gttatatagc ataaatcaat
     1681 attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc
     1741 tcatgtccaa tatgaccgcc atgttggcat tgattattga ctagttatta atagtaatca
     1801 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1861 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1921 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1981 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2041 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2101 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2161 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     2221 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     2281 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     2341 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     2401 ctccatagaa gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg
     2461 cggattcccc gtgccaagag tgacgtaagt accgcctata gagtctatag gcccaccccc
     2521 ttggcttcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     2581 tttaggtgac actatagaat aacatccact ttgcctttct ctccacaggt gtccactccc
     2641 aggtccaact gcacctcggt tctatcgaaa acgcgcctgc tagcgccacc atgaagacca
     2701 tcattgccct gagctacatc ttctgcctgg tgttcgctgc ggccgcccat gaaggcgtgc
     2761 acagaaaacc ttctctgctg gcccatcctg gacctctggt caagagcgaa gagacagtga
     2821 tcctgcagtg ttggagcgac gtgatgttcg agcacttcct gctgcaccgc gagggcatgt
     2881 tcaacgacac actgagactg atcggcgagc accacgatgg cgtgtccaag gccaatttca
     2941 gcatcagcag aatgacccag gacctggccg gcacctacag atgttatggc tctgtgacac
     3001 acagccccta ccaggtgtca gcccctagcg atcctctgga catcgtgatc atcggcctgt
     3061 acgagaagcc tagcctgtct gctcagcctg gacctacagt gctggccggc gaaaatgtga
     3121 ccctgagctg tagcagccgg tccagctacg atatgtacca cctgtccaga gagggcgaag
     3181 cccacgaaag aaggctgcct gccggaccta aagtgaacgg caccttccag gccgattttc
     3241 cactgggacc tgccacacac ggcggcacat atagatgctt cggcagcttc cacgacagcc
     3301 cttacgagtg gtctaagagc agcgatccac tgctggtgtc cgtgaccggc aatcctagca
     3361 atagctggcc ttctccaacc gagcctagca gcaagacagg caaccccaga catctgcatg
     3421 gcgcgccggg cggcgacaag acccacacct gccccccctg cccagctcca gaactgctgg
     3481 gagggccttc cgtgttcctg ttccccccca agcccaagga caccctgatg atcagcagga
     3541 cccccgaggt gacctgcgtg gtggtggccg tgtcccacga ggacccagag gtgaagttca
     3601 actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccaga gaggagcagt
     3661 acaacagcac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg
     3721 gcaaagaata caagtgcaaa gtctccaaca aggccctggc tgccccaatc gaaaagacaa
     3781 tcagcaaggc caagggccag ccacgggagc cccaggtgta caccctgccc cccagccggg
     3841 aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc taccccagcg
     3901 atatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag accacccccc
     3961 cagtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg gacaagtcca
     4021 ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg cacaaccact
     4081 acacccagaa gtccctgagc ctgagccccg gcggcagcgg tcttaatgat attttcgagg
     4141 ctcagaagat tgagtggcat gaacaccacc accatcacca ctaaggccgg cctgtagtca
     4201 ctggcacaac gcgttacccg gtaagccaat cgggtataca cggtcgtcat actgcagaca
     4261 gggttcttct actttgcaag atagtcttga gtagtaaaat aaatagatag agaaaatcag
     4321 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4381 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcac
     4441 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4501 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4561 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4621 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4681 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4741 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4801 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4861 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4921 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     4981 acgacaaggt gaggaactaa accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     5041 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     5101 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     5161 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     5221 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     5281 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     5341 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     5401 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     5461 gcgccggggt gcccgccttc ctggagacat ccgcgccccg caacctcccc ttctacgagc
     5521 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     5581 tgacccgcaa gcccggtgcc tgacacgtgc tacgagattt cgattccacc gccgccttct
     5641 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5701 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5761 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5821 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5881 gctagagctt ggcgtaatca tggtcattac caatgcttaa tcagtgaggc acctatctca
     5941 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6001 atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga accacgctca
     6061 ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6121 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6181 agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca
     6241 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6301 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6361 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6421 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6481 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6541 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6601 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6661 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6721 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt cttccttt
//
