LOCUS       sequence-433194-        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-KIR2DS3-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     132..151
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      237..825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     726..745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     979..996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1094..1111
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1095..1111
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1567..1583
                     /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        1750..2129
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2130..2333
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2284..2304
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2330..2354
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2564..2582
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2564..2581
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2595..2614
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4103..4147
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4148..4165
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4305..4322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4311..4422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4540..4560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4545..4874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4725..4860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4787..4806
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4922..4969
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4988..5587
                     /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(4988..5007)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5484..5504
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5717..5838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5754..5773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5808..5827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5891..6682,6683..6751))
                     /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     6514..6533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6752..6778,1..65))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
       61 acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg cgagcccatt
      121 tatacctgaa tatggctcat aacacccctt gctcatgacc aaaatccctt aacgtgagtt
      181 acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      361 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca ccacttcaag
      421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga gagtagggaa
     1021 ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc gtttctacaa
     1081 actctttctg tgttgtaaaa cgacggccag tcttaagctc gggccccctg ggcggttctg
     1141 ataacgagta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg gtttttttat
     1201 ggggggagtt tagggaaaga gcatttgtca gaatatttaa gggcgcctgt cactttgctt
     1261 gatatatgag aattatttaa ccttataaat gagaaaaaag caacgcactt taaataagat
     1321 acgttgcttt ttcgattgat gaacacctat aattaaacta ttcatctatt atttatgatt
     1381 ttttgtatat acaatatttc tagtttgtta aagagaatta agaaaataaa tctcgaaaat
     1441 aataaaggga aaatcagttt ttgatatcaa aattatacat gtcaacgata atacaaaata
     1501 taatacaaac tataagatgt tatcagtatt tattatcatt tagaataaat tttgtgtcgc
     1561 ccttaattgt gagcggataa caattacgag cttcatgcac agtggcgttg actcaatatt
     1621 ggccattagc catattattc attggttata tagcataaat caatattggc tattggccat
     1681 tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac
     1741 cgccatgttg gcattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     1801 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     1861 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     1921 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     1981 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     2041 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     2101 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     2161 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     2221 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     2281 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
     2341 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
     2401 gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca
     2461 agagtgacgt aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac
     2521 gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg tgacactata
     2581 gaataacatc cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct
     2641 cggttctatc gaaaacgcgc ctgctagcgc caccatgaag accatcattg ccctgagcta
     2701 catcttctgc ctggtgttcg ctgcggccgc ccatgagggc tttagaagaa agccttctct
     2761 gctggctcac cccggcagac tggtcaagtc tgaagagaca gtgatcctgc agtgttggag
     2821 cgacgtgatg ttcgagcact tcctgctgca cagagagggc accttcaacg acacactgag
     2881 actgatcggc gagcacatcg atggcgtgtc caaggccaat ttcagcatcg gcagaatgag
     2941 acaggacctg gccggcacct acagatgtta tggctctgtg cctcacagcc cctaccagtt
     3001 ttctgcccct agcgaccctc tggacatcgt gatcacagga ctgtacgaga agcccagcct
     3061 gtctgctcag cctggaccta cagttctggc cggcgaaagc gtgacactga gctgtagcag
     3121 ctggtccagc tacgacatgt accacctgtc tacagagggc gaagcccacg agagaagatt
     3181 cagcgccgga cctaaagtga acggcacctt ccaggccgat tttccactgg gacctgccac
     3241 acaaggcggc acatatagat gcttcggcag cttccacgac agcccttacg agtggtctaa
     3301 gagcagcgat cctctgctgg tgtccgtgac cggcaatcct agcaatagct ggccttctcc
     3361 aaccgagcct agcagcaaga caggcaaccc cagacatctg catggcgcgc cgggcggcga
     3421 caagacccac acctgccccc cctgcccagc tccagaactg ctgggagggc cttccgtgtt
     3481 cctgttcccc cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg
     3541 cgtggtggtg gccgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg
     3601 cgtggaggtg cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag
     3661 ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg aacggcaaag aatacaagtg
     3721 caaagtctcc aacaaggccc tggctgcccc aatcgaaaag acaatcagca aggccaaggg
     3781 ccagccacgg gagccccagg tgtacaccct gccccccagc cgggaggaga tgaccaagaa
     3841 ccaggtgtcc ctgacctgtc tggtgaaggg cttctacccc agcgatatcg ccgtggagtg
     3901 ggagagcaac ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga
     3961 cggcagcttc ttcctgtaca gcaagctgac cgtggacaag tccaggtggc agcagggcaa
     4021 cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct
     4081 gagcctgagc cccggcggca gcggtcttaa tgatattttc gaggctcaga agattgagtg
     4141 gcatgaacac caccaccatc accactaagg ccggcctgta gtcactggca caacgcgtta
     4201 cccggtaagc caatcgggta tacacggtcg tcatactgca gacagggttc ttctactttg
     4261 caagatagtc ttgagtagta aaataaatag atagagaaaa tcagcctcga ctgtgccttc
     4321 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4381 cactcccact gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc
     4441 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4501 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     4561 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4621 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4681 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4741 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4801 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4861 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     4921 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     4981 ctaaaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     5041 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     5101 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     5161 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     5221 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     5281 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     5341 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     5401 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     5461 cttcctggag acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     5521 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     5581 tgcctgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5641 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5701 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5761 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5821 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5881 atcatggtca ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6001 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     6061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6181 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6721 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaag
//
