LOCUS       sequence-302857-        7522 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 7522)
  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 7522)
  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-MUSK-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7522
                     /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             4864..4908
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4909..4926
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5066..5083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5072..5183
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5301..5321)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5306..5635
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5486..5621
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5548..5567
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5683..5730
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5749..6348
                     /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(5749..5768)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6245..6265
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6478..6599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6515..6534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6569..6588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6652..7443,7444..7512))
                     /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     7275..7294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7513..7522,1..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 cggccgcctt gcctaaggct
     2761 cctgtgatta ccacacctct ggaaaccgtg gacgccctgg tggaagaggt ggccactttt
     2821 atgtgcgccg tggaaagcta ccctcagcct gagatcagct ggacccggaa caagatcctg
     2881 atcaagctgt tcgacacccg gtacagcatc agagagaatg gccagctgct gaccatcctg
     2941 agcgtggaag atagcgacga cggcatctac tgctgcaccg ccaacaatgg tgttggcgga
     3001 gctgtggaat cttgtggcgc cctgcaagtg aagatgaagc ccaagatcac ccggcctcca
     3061 atcaacgtga agatcatcga gggactgaag gccgtgctgc cttgcaccac aatgggcaat
     3121 cctaagccta gcgtgtcctg gatcaagggc gatagccctc tgagagagaa cagcagaatc
     3181 gccgtgctgg aaagcggctc cctgagaatc cacaacgtgc agaaagagga cgccggccag
     3241 tacagatgcg tggccaagaa ttctctgggc accgcctaca gcaaggtggt caagctggaa
     3301 gtggaagtgt tcgccagaat cctgagagcc cctgagtctc acaatgtgac cttcggcagc
     3361 ttcgtgaccc tgcactgtac agccacaggc atccctgtgc ctaccatcac ctggatcgag
     3421 aacggcaatg ccgtgtccag cggcagcatc caagagtccg tgaaggacag agtgatcgac
     3481 agcagactgc agctgttcat caccaagcct ggcctgtaca cctgtatcgc caccaacaag
     3541 cacggggaga agttctctac agccaaggcc gctgccacca tctctatcgc cgaatggtcc
     3601 aagcctcaga aggacaacaa gggctactgc gcccagtatc ggggcgaagt ctgtaatgct
     3661 gtgctggcca aggatgccct ggtgttcctg aataccagct acgccgatcc tgaagaggcc
     3721 caagaactgc tggtgcacac agcctggaat gagctgaagg tggtgtcccc tgtgtgtaga
     3781 cctgctgctg aggccctgct gtgcaaccac atcttccaag agtgtagccc tggcgtggtg
     3841 cccacaccta ttcctatctg cagagaatac tgcctggccg tgaaagagct gttctgcgcc
     3901 aaagaatggc tggtcatgga agagaaaacc caccggggcc tgtatcggag cgaaatgcat
     3961 ctcctgtccg tgcctgagtg cagcaagctg ccttctatgc actgggaccc tacagcctgt
     4021 gccagactgc cacacctgga ctacaacaaa gagaacctca agacctttcc accgatgacc
     4081 agcagcaagc ccagcgtgga catccctaat ctgcctagca gcagcagctc cagcttcagc
     4141 gtgtccccta cctacagcat gacaggcgcg ccgggcggcg acaagaccca cacctgcccc
     4201 ccctgcccag ctccagaact gctgggaggg ccttccgtgt tcctgttccc ccccaagccc
     4261 aaggacaccc tgatgatcag caggaccccc gaggtgacct gcgtggtggt ggccgtgtcc
     4321 cacgaggacc cagaggtgaa gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc
     4381 aagaccaagc ccagagagga gcagtacaac agcacctaca gggtggtgtc cgtgctgacc
     4441 gtgctgcacc aggactggct gaacggcaaa gaatacaagt gcaaagtctc caacaaggcc
     4501 ctggctgccc caatcgaaaa gacaatcagc aaggccaagg gccagccacg ggagccccag
     4561 gtgtacaccc tgccccccag ccgggaggag atgaccaaga accaggtgtc cctgacctgt
     4621 ctggtgaagg gcttctaccc cagcgatatc gccgtggagt gggagagcaa cggccagccc
     4681 gagaacaact acaagaccac ccccccagtg ctggacagcg acggcagctt cttcctgtac
     4741 agcaagctga ccgtggacaa gtccaggtgg cagcagggca acgtgttcag ctgcagcgtg
     4801 atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgagcctgag ccccggcggc
     4861 agcggtctta atgatatttt cgaggctcag aagattgagt ggcatgaaca ccaccaccat
     4921 caccactaag gccggcctgt agtcactggc acaacgcgtt acccggtaag ccaatcgggt
     4981 atacacggtc gtcatactgc agacagggtt cttctacttt gcaagatagt cttgagtagt
     5041 aaaataaata gatagagaaa atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5101 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5161 taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat tcttttgatt
     5221 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5281 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     5341 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     5401 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5461 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5521 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     5581 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     5641 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
     5701 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gaccgagtac
     5761 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     5821 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     5881 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     5941 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     6001 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     6061 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     6121 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     6181 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     6241 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     6301 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga
     6361 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     6421 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     6481 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     6541 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     6601 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc attaccaatg
     6661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     6781 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     6841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6961 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7261 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7501 ttgaatactc atattcttcc tt
//
