LOCUS       sequence-302325-        6694 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 6694)
  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 6694)
  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-TMEM81-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6694
                     /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             4036..4080
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4081..4098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4238..4255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4244..4355
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4473..4493)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4478..4807
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4658..4793
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4720..4739
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4855..4902
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4921..5520
                     /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(4921..4940)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5417..5437
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5650..5771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5687..5706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5741..5760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5824..6615,6616..6684))
                     /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     6447..6466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6685..6694,1..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 cggccgccat tccagaaaag
     2761 ctgcaagagg ccgtgggcaa agtgatcatc aacgccacca cctgtaccgt gacatgcggc
     2821 ctgggctaca aagaagagac agtctgcgaa gtgggacccg acggcgttag aagaaagtgt
     2881 cagaccagac ggctggaatg cctgaccaac tggatctgcg gcatgctgca cttcaccatc
     2941 ctgatcggca aagagttcga gctgagctgc ctgagcagcg acatcctgga attcggccaa
     3001 gaggccttca gattcacatg gcgactggcc agaggcgtga tcagcaccga tgatgaggtg
     3061 ttcaagccct tccaggccaa cagccacttc gtgaagttta agtacgccca agagtacgac
     3121 agcggcacct acagatgtga cgtgcagctg gtcaagaacc tgagactggt caagcggctg
     3181 tacttcggcc tgagagtgct gcctcctaac ctggtcaacc tgaacttcca ccagagcctg
     3241 accgaggacc agaagctgat cgacgaagga ctggaagtga acctggacag ctacagcaag
     3301 ccccaccatc ctaagtggaa gaaaaaggtg gcctctggcg cgccgggcgg cgacaagacc
     3361 cacacctgcc ccccctgccc agctccagaa ctgctgggag ggccttccgt gttcctgttc
     3421 ccccccaagc ccaaggacac cctgatgatc agcaggaccc ccgaggtgac ctgcgtggtg
     3481 gtggccgtgt cccacgagga cccagaggtg aagttcaact ggtacgtgga cggcgtggag
     3541 gtgcacaacg ccaagaccaa gcccagagag gagcagtaca acagcaccta cagggtggtg
     3601 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagaatacaa gtgcaaagtc
     3661 tccaacaagg ccctggctgc cccaatcgaa aagacaatca gcaaggccaa gggccagcca
     3721 cgggagcccc aggtgtacac cctgcccccc agccgggagg agatgaccaa gaaccaggtg
     3781 tccctgacct gtctggtgaa gggcttctac cccagcgata tcgccgtgga gtgggagagc
     3841 aacggccagc ccgagaacaa ctacaagacc acccccccag tgctggacag cgacggcagc
     3901 ttcttcctgt acagcaagct gaccgtggac aagtccaggt ggcagcaggg caacgtgttc
     3961 agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg
     4021 agccccggcg gcagcggtct taatgatatt ttcgaggctc agaagattga gtggcatgaa
     4081 caccaccacc atcaccacta aggccggcct gtagtcactg gcacaacgcg ttacccggta
     4141 agccaatcgg gtatacacgg tcgtcatact gcagacaggg ttcttctact ttgcaagata
     4201 gtcttgagta gtaaaataaa tagatagaga aaatcagcct cgactgtgcc ttctagttgc
     4261 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4321 actgtccttt cctaataaaa tgaggaaatt gcatcacaac actcaaccct atctcggtct
     4381 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4441 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     4501 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4561 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4621 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4681 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4741 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4801 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     4861 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     4921 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     4981 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     5041 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     5101 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     5161 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     5221 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     5281 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     5341 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     5401 gagacatccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     5461 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     5521 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5581 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5641 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5701 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5761 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5821 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5881 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5941 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     6001 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6061 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6121 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6181 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6241 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6301 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6361 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6421 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6481 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6541 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6601 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6661 gacacggaaa tgttgaatac tcatattctt cctt
//
