LOCUS       sequence-302577-        6988 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 6988)
  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 6988)
  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-NRG2-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6988
                     /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             4330..4374
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4375..4392
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4532..4549)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4538..4649
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4767..4787)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4772..5101
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4952..5087
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5014..5033
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5149..5196
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5215..5814
                     /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(5215..5234)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5711..5731
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5944..6065
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5981..6000)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6035..6054
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6118..6909,6910..6978))
                     /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     6741..6760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6979..6988,1..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 cggccgcctg ttactctcct
     2761 agcctgaagt ctgtgcagga ccaggcctat aaggcccctg tggtggtgga aggcaaagtg
     2821 caaggacttg tgcctgccgg cggaagcagc agcaattcta caagagaacc tcctgccagc
     2881 ggcagagtgg ctctggttaa ggttctggac aagtggcctc tgagatctgg cggcctgcag
     2941 agggaacaag tgatctctgt gggcagctgc gtgcccctgg aacggaacca gcggtacatc
     3001 ttctttctgg aacccaccga gcagcccctg gtgttcaaaa cagccttcgc tcccctggac
     3061 accaacggca agaacctgaa gaaagaagtg ggcaagatcc tctgcaccga ctgcgccaca
     3121 agacccaagc tgaagaagat gaagtcccag accggccaag tgggcgagaa gcagtctctg
     3181 aaatgtgaag ccgctgccgg caatccccag cctagctaca gatggttcaa ggacggcaaa
     3241 gagctgaaca gaagccggga catccggatt aagtacggca acggccggaa gaacagcagg
     3301 ctgcagttca acaaagtgaa ggtggaagat gccggcgagt acgtgtgcga ggccgagaat
     3361 atcctgggca aagatacagt gcggggcaga ctgtacgtga acagcgtgtc cacaacactg
     3421 tcctcttgga gcggccacgc cagaaagtgc aatgagacag ccaagagcta ctgcgtgaac
     3481 ggcggcgtgt gttactacat cgagggcatc aaccagctga gctgcaagtg ccctaatggc
     3541 ttcttcggcc agcggtgcct ggaaaagctg cccctgagac tgtacatgcc cgatcctaag
     3601 cagaaggccg aggaactgta ccagaaacga ggcgcgccgg gcggcgacaa gacccacacc
     3661 tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct gttccccccc
     3721 aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggcc
     3781 gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac
     3841 aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg
     3901 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa agtctccaac
     3961 aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca gccacgggag
     4021 ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg
     4081 acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga gagcaacggc
     4141 cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc
     4201 ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt gttcagctgc
     4261 agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcccc
     4321 ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca tgaacaccac
     4381 caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa
     4441 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg
     4501 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca
     4561 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4621 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt
     4681 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4741 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4801 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4861 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4921 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4981 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     5041 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     5101 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     5161 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc
     5221 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     5281 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     5341 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     5401 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     5461 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     5521 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     5581 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     5641 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca
     5701 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     5761 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg
     5821 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5881 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5941 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6001 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6061 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta
     6121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6241 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
     6301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6421 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6601 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6661 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6721 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6781 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6841 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6901 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6961 gaaatgttga atactcatat tcttcctt
//
