LOCUS       sequence-302503-        6835 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 6835)
  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 6835)
  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-TNFSF10-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     149..168
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      254..842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     743..762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     996..1013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1111..1128
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1112..1128
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1584..1600
                     /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        1767..2146
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2147..2350
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2301..2321
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2347..2371
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2581..2599
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2581..2598
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2612..2631
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4177..4221
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4222..4239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4379..4396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4385..4496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4614..4634)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4619..4948
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4799..4934
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4861..4880
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4996..5043
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5062..5661
                     /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(5062..5081)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5558..5578
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5791..5912
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5828..5847)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5882..5901
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5965..6756,6757..6825))
                     /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     6588..6607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6826..6835,1..82))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac
      121 attatcgcga gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa
      181 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
      241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      361 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      421 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     1021 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     1081 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg
     1141 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct
     1201 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg
     1261 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa
     1321 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc
     1381 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga
     1441 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc
     1501 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag
     1561 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt
     1621 ggcgttgact caatattggc cattagccat attattcatt ggttatatag cataaatcaa
     1681 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg
     1741 ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc
     1801 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1861 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1921 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1981 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2041 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2101 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2161 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2221 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2281 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2341 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     2401 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac
     2461 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc
     2521 cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     2581 atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc
     2641 caggtccaac tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc
     2701 atcattgccc tgagctacat cttctgcctg gtgttcgctg cggccgccac aaatgagctg
     2761 aagcagatgc aggacaagta cagcaagagc ggaatcgcct gcttcctgaa agaggacgac
     2821 agctactggg accccaacga cgaagagagc atgaacagcc cctgctggca agtgaagtgg
     2881 cagctgagac agctcgtgcg gaagatgatc ctgcggacca gcgaagagac aatcagcacc
     2941 gtgcaagaga agcagcagaa catcagccct ctcgtcagag agagaggccc tcagagagtt
     3001 gccgctcaca ttaccggcac cagaggcaga agcaataccc tgagcagccc caacagcaag
     3061 aatgagaagg ccctgggcag aaagatcaac agctgggagt ctagcagaag cggccacagc
     3121 tttctgagca acctgcacct gagaaacggc gagctggtta tccacgagaa gggcttctac
     3181 tacatctaca gccagaccta cttccggttc caagaggaaa tcaaagagaa caccaagaac
     3241 gacaagcaga tggtgcagta catctataag tacacgagct accccgatcc gatcctgctg
     3301 atgaagtccg ccagaaacag ctgctggtcc aaggatgccg agtacggcct gtacagcatc
     3361 taccaaggcg gcatcttcga gctgaaagag aatgaccgga tcttcgtgtc cgtgaccaac
     3421 gagcacctga tcgacatgga ccacgaggcc agctttttcg gcgcctttct cgttggaggc
     3481 gcgccgggcg gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga
     3541 gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc
     3601 cccgaggtga cctgcgtggt ggtggccgtg tcccacgagg acccagaggt gaagttcaac
     3661 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac
     3721 aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc
     3781 aaagaataca agtgcaaagt ctccaacaag gccctggctg ccccaatcga aaagacaatc
     3841 agcaaggcca agggccagcc acgggagccc caggtgtaca ccctgccccc cagccgggag
     3901 gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat
     3961 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca
     4021 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagtccagg
     4081 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
     4141 acccagaagt ccctgagcct gagccccggc ggcagcggtc ttaatgatat tttcgaggct
     4201 cagaagattg agtggcatga acaccaccac catcaccact aaggccggcc tgtagtcact
     4261 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg
     4321 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc
     4381 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4441 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa
     4501 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4561 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     4621 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4681 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4741 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4801 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4861 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     4921 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     4981 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     5041 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     5101 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     5161 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     5221 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     5281 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     5341 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     5401 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     5461 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     5521 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg
     5581 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     5641 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat
     5701 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5761 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5821 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5881 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5941 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc
     6001 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6061 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     6121 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6181 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6241 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     6301 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6361 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6421 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6481 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6541 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6601 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6661 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6721 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6781 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt
//
