LOCUS       sequence-368600-        6514 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 6514)
  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 6514)
  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-KLRG1-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     105..124
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(143..190)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(238..567)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      252..387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(306..325)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     552..572
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    690..801
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     790..807
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(947..964)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(965..1009)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2234..2253)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2266..2284)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2267..2284)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2494..2518)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2515..2718)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2544..2564)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2719..3098
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    3265..3281
                     /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)."
     primer_bind     complement(3737..3754)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3737..3753)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3852..3869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4023..4611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4103..4122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4697..4716)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4722..4741)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4783..4874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4875..4943,4944..5735)
                     /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     complement(5093..5112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    5788..5909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5799..5818)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5853..5872
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6039..6514,1..124))
                     /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(6122..6142)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg
       61 tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg
      121 tcatggttta gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt
      181 aattgtcaac acgtgctgat cagatccgaa aatggatata caagctcccg ggagcttttt
      241 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga
      301 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
      361 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
      421 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
      481 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      541 ggactttcca caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt
      601 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      661 gaatagaccg agatagggtt gagtgttgtg atgcaatttc ctcattttat taggaaagga
      721 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
      781 gctggcaact agaaggcaca gtcgaggctg attttctcta tctatttatt ttactactca
      841 agactatctt gcaaagtaga agaaccctgt ctgcagtatg acgaccgtgt atacccgatt
      901 ggcttaccgg gtaacgcgtt gtgccagtga ctacaggccg gccttagtgg tgatggtggt
      961 ggtgttcatg ccactcaatc ttctgagcct cgaaaatatc attaagaccg ctgccgccgg
     1021 ggctcaggct cagggacttc tgggtgtagt ggttgtgcag ggcctcgtgc atcacgctgc
     1081 agctgaacac gttgccctgc tgccacctgg acttgtccac ggtcagcttg ctgtacagga
     1141 agaagctgcc gtcgctgtcc agcactgggg gggtggtctt gtagttgttc tcgggctggc
     1201 cgttgctctc ccactccacg gcgatatcgc tggggtagaa gcccttcacc agacaggtca
     1261 gggacacctg gttcttggtc atctcctccc ggctgggggg cagggtgtac acctggggct
     1321 cccgtggctg gcccttggcc ttgctgattg tcttttcgat tggggcagcc agggccttgt
     1381 tggagacttt gcacttgtat tctttgccgt tcagccagtc ctggtgcagc acggtcagca
     1441 cggacaccac cctgtaggtg ctgttgtact gctcctctct gggcttggtc ttggcgttgt
     1501 gcacctccac gccgtccacg taccagttga acttcacctc tgggtcctcg tgggacacgg
     1561 ccaccaccac gcaggtcacc tcgggggtcc tgctgatcat cagggtgtcc ttgggcttgg
     1621 gggggaacag gaacacggaa ggccctccca gcagttctgg agctgggcag ggggggcagg
     1681 tgtgggtctt gtcgccgccc ggcgcgccaa acagggcctg atcggcaaat gggcacttct
     1741 tgcagaccca gtgaagtggc acctcgcagc tgctggcctg cagtccattt ttgttgatgg
     1801 cgccgcaggt ctgcacgaag ctgttgctgc tgattctgct gaagttcaga gggctgccgt
     1861 cctcccatct ccaaccgcta ttgtttctca ggccgatcca acaaaaggcc tcgctcagga
     1921 acacctggag caggctcatc tcttgattgt cggtgatgac cagcagatgg ctgtctctgg
     1981 ccaggcagaa ttccaggctg ctgttccagt ccttctcttc cacgctgaag tagtagcagt
     2041 ggttgccgta cttcatccat ctgtcggggc agctaggaca gctggcgcag gtagagtaat
     2101 tgctgccctg acacaaggcg gccgcagcga acaccaggca gaagatgtag ctcagggcaa
     2161 tgatggtctt catggtggcg ctagcaggcg cgttttcgat agaaccgagg tgcagttgga
     2221 cctgggagtg gacacctgtg gagagaaagg caaagtggat gttattctat agtgtcacct
     2281 aaatcgtatg tgtatgatac ataaggttat gtattaattg tagccgcgtt ctaacgaagc
     2341 caagggggtg ggcctataga ctctataggc ggtacttacg tcactcttgg cacggggaat
     2401 ccgcgttcca atgcaccgtt cccggccgcg gaggctggat cggtcccggt gtcttctatg
     2461 gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga cggttcacta aacgagctct
     2521 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt
     2581 gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     2641 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta
     2701 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     2761 aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     2821 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     2881 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     2941 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     3001 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     3061 aattgattac tattaataac tagtcaataa tcaatgccaa catggcggtc atattggaca
     3121 tgagccaata taaatgtaca tattatgata tagatacaac gtatgcaatg gccaatagcc
     3181 aatattgatt tatgctatat aaccaatgaa taatatggct aatggccaat attgagtcaa
     3241 cgccactgtg catgaagctc gtaattgtta tccgctcaca attaagggcg acacaaaatt
     3301 tattctaaat gataataaat actgataaca tcttatagtt tgtattatat tttgtattat
     3361 cgttgacatg tataattttg atatcaaaaa ctgattttcc ctttattatt ttcgagattt
     3421 attttcttaa ttctctttaa caaactagaa atattgtata tacaaaaaat cataaataat
     3481 agatgaatag tttaattata ggtgttcatc aatcgaaaaa gcaacgtatc ttatttaaag
     3541 tgcgttgctt ttttctcatt tataaggtta aataattctc atatatcaag caaagtgaca
     3601 ggcgccctta aatattctga caaatgctct ttccctaaac tccccccata aaaaaacccg
     3661 ccgaagcggg tttttacgtt atttgcggat taacgattac tcgttatcag aaccgcccag
     3721 ggggcccgag cttaagactg gccgtcgttt tacaacacag aaagagtttg tagaaacgca
     3781 aaaaggccat ccgtcagggg ccttctgctt agtttgatgc ctggcagttc cctactctcg
     3841 ccttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4441 gctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4621 ttgatctttt ctacggggtc tgacgctcag tggaacgacg cgcgcgtaac tcacgttaag
     4681 ggattttggt catgagcaag gggtgttatg agccatattc aggtataaat gggctcgcga
     4741 taatgttcag aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa
     4801 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4861 gaaaaaggaa gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4921 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4981 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5041 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5101 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5161 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5221 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5281 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5341 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5401 gtgacaccac gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac
     5461 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5521 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg
     5581 gtgagcgtgg ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta
     5641 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5701 ctgagatagg tgcctcactg attaagcatt ggtaatgacc atgattacgc caagctctag
     5761 ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa
     5821 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     5881 taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc
     5941 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     6001 tagaaggcgg cggtggaatc gaaatctcgt agcacgtgtc aggcaccggg cttgcgggtc
     6061 atgcaccagg tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc
     6121 cgctcgtaga aggggaggtt gcggggcgcg gatgtctcca ggaaggcggg caccccggcg
     6181 cgctcggccg cctccactcc ggggagcacg acggcgctgc ccagaccctt gccctggtgg
     6241 tcgggcgaga cgccgacggt ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc
     6301 aggaggcctt ccatctgttg ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc
     6361 gggccgatct cggcgaacac cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc
     6421 accgcggcgc cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg
     6481 aagagttctt gcagctcggt gacccgctcg atgt
//
