LOCUS       sequence-368388-        6265 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 6265)
  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 6265)
  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-TNFRSF12A-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     371..391
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    604..725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(641..660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     695..714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(778..1569,1570..1638))
                     /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     1401..1420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1639..1730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1772..1791
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1797..1816
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1902..2490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2391..2410
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2644..2661
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2759..2776
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2760..2776
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3232..3248
                     /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        3415..3794
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3795..3998
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3949..3969
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3995..4019
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        4229..4247
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4229..4246
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     4260..4279
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             5255..5299
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5300..5317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5457..5474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5463..5574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5692..5712)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5697..6026
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5877..6012
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5939..5958
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6074..6121
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(6140..6265,1..474)
                     /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(6140..6159)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
       61 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
      121 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
      181 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
      241 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
      301 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca
      361 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
      421 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg
      481 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
      541 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
      601 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      661 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      721 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta
      781 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      841 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      901 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
      961 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1021 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1081 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1141 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1201 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1261 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1321 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1381 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     1441 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     1501 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1561 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1621 gaaatgttga atactcatat tcttcctttt tcaatattat tgaagcattt atcagggtta
     1681 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggtcag
     1741 tgttacaacc aattaaccaa ttctgaacat tatcgcgagc ccatttatac ctgaatatgg
     1801 ctcataacac cccttgctca tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc
     1861 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1921 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1981 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2041 aatactgttc ttctagtgta gccgtagtta gcccaccact tcaagaactc tgtagcaccg
     2101 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2161 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2221 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2281 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2341 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2401 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2461 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2521 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2581 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2641 cgcagcgagt cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa
     2701 ctaagcagaa ggcccctgac ggatggcctt tttgcgtttc tacaaactct ttctgtgttg
     2761 taaaacgacg gccagtctta agctcgggcc ccctgggcgg ttctgataac gagtaatcgt
     2821 taatccgcaa ataacgtaaa aacccgcttc ggcgggtttt tttatggggg gagtttaggg
     2881 aaagagcatt tgtcagaata tttaagggcg cctgtcactt tgcttgatat atgagaatta
     2941 tttaacctta taaatgagaa aaaagcaacg cactttaaat aagatacgtt gctttttcga
     3001 ttgatgaaca cctataatta aactattcat ctattattta tgattttttg tatatacaat
     3061 atttctagtt tgttaaagag aattaagaaa ataaatctcg aaaataataa agggaaaatc
     3121 agtttttgat atcaaaatta tacatgtcaa cgataataca aaatataata caaactataa
     3181 gatgttatca gtatttatta tcatttagaa taaattttgt gtcgccctta attgtgagcg
     3241 gataacaatt acgagcttca tgcacagtgg cgttgactca atattggcca ttagccatat
     3301 tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc
     3361 tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt
     3421 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     3481 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     3541 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     3601 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     3661 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     3721 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     3781 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     3841 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     3901 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     3961 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     4021 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     4081 tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta
     4141 ccgcctatag agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat
     4201 acataacctt atgtatcata cacatacgat ttaggtgaca ctatagaata acatccactt
     4261 tgcctttctc tccacaggtg tccactccca ggtccaactg cacctcggtt ctatcgaaaa
     4321 cgcgcctgct agcgccacca tgaagaccat cattgccctg agctacatct tctgcctggt
     4381 gttcgctgcg gccgccgaac aagctcctgg aacagctcct tgtagcagag gaagctcttg
     4441 gagcgccgac ctggacaagt gtatggattg cgccagctgc agagcccggc ctcactctga
     4501 tttttgtctg ggatgtgccg ccgctcctcc tgctcctttt agactgcttt ggcctggcgc
     4561 gccgggcggc gacaagaccc acacctgccc cccctgccca gctccagaac tgctgggagg
     4621 gccttccgtg ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc
     4681 cgaggtgacc tgcgtggtgg tggccgtgtc ccacgaggac ccagaggtga agttcaactg
     4741 gtacgtggac ggcgtggagg tgcacaacgc caagaccaag cccagagagg agcagtacaa
     4801 cagcacctac agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa
     4861 agaatacaag tgcaaagtct ccaacaaggc cctggctgcc ccaatcgaaa agacaatcag
     4921 caaggccaag ggccagccac gggagcccca ggtgtacacc ctgcccccca gccgggagga
     4981 gatgaccaag aaccaggtgt ccctgacctg tctggtgaag ggcttctacc ccagcgatat
     5041 cgccgtggag tgggagagca acggccagcc cgagaacaac tacaagacca cccccccagt
     5101 gctggacagc gacggcagct tcttcctgta cagcaagctg accgtggaca agtccaggtg
     5161 gcagcagggc aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac
     5221 ccagaagtcc ctgagcctga gccccggcgg cagcggtctt aatgatattt tcgaggctca
     5281 gaagattgag tggcatgaac accaccacca tcaccactaa ggccggcctg tagtcactgg
     5341 cacaacgcgt tacccggtaa gccaatcggg tatacacggt cgtcatactg cagacagggt
     5401 tcttctactt tgcaagatag tcttgagtag taaaataaat agatagagaa aatcagcctc
     5461 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5521 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca
     5581 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5641 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     5701 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5761 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     5821 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     5881 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5941 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     6001 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     6061 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     6121 caaggtgagg aactaaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     6181 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     6241 ccacaccgtc gatccggacc gccac
//
