LOCUS       sequence-433181-        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-KIR2DL3-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     114..133
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     139..158
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      244..832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     733..752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     986..1003
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1101..1118
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1102..1118
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1574..1590
                     /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        1757..2136
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2137..2340
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2291..2311
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2337..2361
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2571..2589
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2571..2588
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2602..2621
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4110..4154
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4155..4172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4312..4329)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4318..4429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4547..4567)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4552..4881
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4732..4867
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4794..4813
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4929..4976
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4995..5594
                     /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(4995..5014)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5491..5511
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5724..5845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5761..5780)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5815..5834
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5898..6689,6690..6758))
                     /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     6521..6540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6759..6778,1..72))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
       61 aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga
      121 gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa atcccttaac
      181 gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      241 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      301 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      361 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt tagcccacca
      421 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      481 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      541 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
      601 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
      661 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      721 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      781 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      841 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      901 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      961 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaggcgagag
     1021 tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc tttttgcgtt
     1081 tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg ccccctgggc
     1141 ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct tcggcgggtt
     1201 tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg cgcctgtcac
     1261 tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa cgcactttaa
     1321 ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc atctattatt
     1381 tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga aaataaatct
     1441 cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc aacgataata
     1501 caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag aataaatttt
     1561 gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt ggcgttgact
     1621 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat
     1681 tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca
     1741 atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc aattacgggg
     1801 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1861 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1921 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1981 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     2041 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     2101 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     2161 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     2221 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     2281 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     2341 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
     2401 agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc
     2461 cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttcg
     2521 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
     2581 cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac
     2641 tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc atcattgccc
     2701 tgagctacat cttctgcctg gtgttcgctg cggccgccca tgaaggcgtg cacagaaaac
     2761 cttctctgct ggcccatcct ggacctctgg tcaagagcga agagacagtg atcctgcagt
     2821 gttggagcga cgtgcggttc cagcactttc tgctgcacag agagggcaag ttcaaggaca
     2881 cactgcacct gatcggagag caccacgatg gcgtgtccaa ggccaatttc agcatcggcc
     2941 ccatgatgca ggatctggcc ggcacataca gatgctacgg ctctgtgaca cacagcccct
     3001 atcagctgtc tgcccctagc gatcccctgg atatcgtgat caccggcctg tacgagaagc
     3061 ctagcctgtc tgctcagcct ggacctacag ttctggctgg cgaaagcgtg accctgagct
     3121 gtagcagcag atccagctac gacatgtacc acctgtccag agaaggcgag gcccacgaga
     3181 gaagattcag cgccggacct aaagtgaacg gcaccttcca ggccgatttt ccactgggac
     3241 ctgccacaca cggcggcacc tatagatgct ttggcagctt cagagacagc ccttacgagt
     3301 ggtccaacag cagcgatcct ctgctggtgt ctgtgaccgg caatcccagc aatagctggc
     3361 cttctccaac cgagccaagc agcgagacag gcaaccccag acatctgcat ggcgcgccgg
     3421 gcggcgacaa gacccacacc tgccccccct gcccagctcc agaactgctg ggagggcctt
     3481 ccgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcagg acccccgagg
     3541 tgacctgcgt ggtggtggcc gtgtcccacg aggacccaga ggtgaagttc aactggtacg
     3601 tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag tacaacagca
     3661 cctacagggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat
     3721 acaagtgcaa agtctccaac aaggccctgg ctgccccaat cgaaaagaca atcagcaagg
     3781 ccaagggcca gccacgggag ccccaggtgt acaccctgcc ccccagccgg gaggagatga
     3841 ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctaccccagc gatatcgccg
     3901 tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc ccagtgctgg
     3961 acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc aggtggcagc
     4021 agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga
     4081 agtccctgag cctgagcccc ggcggcagcg gtcttaatga tattttcgag gctcagaaga
     4141 ttgagtggca tgaacaccac caccatcacc actaaggccg gcctgtagtc actggcacaa
     4201 cgcgttaccc ggtaagccaa tcgggtatac acggtcgtca tactgcagac agggttcttc
     4261 tactttgcaa gatagtcttg agtagtaaaa taaatagata gagaaaatca gcctcgactg
     4321 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4381 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatca caacactcaa
     4441 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     4561 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4621 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4681 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4741 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4801 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4861 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4921 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     4981 tgaggaacta aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     5041 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5101 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5161 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5221 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5281 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5341 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     5401 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5461 tgcccgcctt cctggagaca tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5521 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5581 agcccggtgc ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     5641 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5701 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5761 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5821 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5881 tggcgtaatc atggtcatta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5941 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6001 ggcttaccat ctggccccag cgctgcgatg ataccgcgag aaccacgctc accggctccg
     6061 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6121 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6181 gttaatagtt tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg
     6241 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6301 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6361 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6421 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6481 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6541 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6601 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6661 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6721 aagggaataa gggcgacacg gaaatgttga atactcatat tcttcctttt tcaatatt
//
