LOCUS       sequence-303077-        8560 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 8560)
  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 8560)
  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-LEPR-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8560
                     /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             5902..5946
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5947..5964
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6104..6121)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6110..6221
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6339..6359)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6344..6673
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6524..6659
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6586..6605
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6721..6768
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6787..7386
                     /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(6787..6806)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7283..7303
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7516..7637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7553..7572)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7607..7626
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7690..8481,8482..8550))
                     /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     8313..8332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8551..8560,1..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 taatctgagc
     2761 taccccatca caccctggcg gttcaagctg agctgcatgc ctcctaactc tacctacgac
     2821 tacttcctgc tgcctgccgg cctgagcaag aacaccagca atagcaacgg ccactacgag
     2881 acagccgtgg aacccaagtt caacagcagc ggcacccact tcagcaacct gtccaagacc
     2941 accttccact gctgcttcag atccgagcag gaccggaatt gcagcctgtg cgccgataac
     3001 atcgagggca agaccttcgt gtccaccgtg aacagcctgg tgttccagca gatcgacgcc
     3061 aactggaata tccagtgctg gctgaagggc gacctgaagc tgttcatctg ctacgtggaa
     3121 agcctgttca agaacctctt ccggaactac aactacaagg tccacctcct gtacgtgctg
     3181 cccgaggtgc tggaagattc tccactggtg cctcagaaag gcagcttcca gatggtgcac
     3241 tgcaactgca gcgtgcacga gtgctgcgaa tgtctggtgc ctgtgcctac cgccaagctg
     3301 aacgataccc tgctgatgtg cctgaagatc acctctggcg gcgtgatctt tcagagccct
     3361 ctgatgagcg tgcagcccat caacatggtc aagcccgatc ctccactggg actgcacatg
     3421 gaaatcaccg acgacggcaa tctgaagatc tcttggagca gccctcctct ggtgcccttt
     3481 ccactgcaat accaagtgaa gtacagcgag aacagcacca ccgtgatccg cgaggccgat
     3541 aagatcgtgt ctgccacaag cctgctggtg gactctatcc tgcctggcag cagctacgag
     3601 gtgcaagtgc ggggcaaaag actggacggc cctggcattt ggagcgattg gagcacccct
     3661 agagtgttca cgacccagga cgtgatctac ttcccaccta agatcctgac cagcgtgggc
     3721 agcaacgtgt cctttcactg catctacaag aaagagaaca agatcgtccc cagcaaagaa
     3781 atcgtgtggt ggatgaacct ggccgagaag atccctcaga gccagtacga cgtggtgtcc
     3841 gaccacgtgt ccaaagtgac cttcttcaac ctgaacgaga caaagcccag aggcaagttc
     3901 acctacgacg ccgtgtactg ctgcaacgag cacgaatgcc accacagata cgccgagctg
     3961 tacgtgatcg acgtgaacat caacatcagc tgcgagacag acggctacct gaccaagatg
     4021 acctgccggt ggtctaccag caccatccag tctctggccg agtctaccct gcagctgaga
     4081 taccacagaa gcagcctgta ctgcagcgac atccccagca ttcaccccat cagcgagccc
     4141 aaggattgct acctgcagag cgacggcttc tacgagtgta tctttcagcc catctttctg
     4201 ctgagcggct acaccatgtg gatcagaatc aaccacagcc tgggcagcct ggactcccct
     4261 cctacatgtg tgctgcctga cagcgtcgtg aagcctctgc ctccatcttc tgtgaaggcc
     4321 gagatcacca tcaatatcgg cctgctgaag attagctggg agaagcctgt gttccccgag
     4381 aacaacctgc agttccagat cagatacggc ctgtccggca aagaggtcca gtggaagatg
     4441 tacgaggtgt acgacgccaa gagcaagagc gtgtcactgc ccgtgcctga tctgtgtgcc
     4501 gtgtatgcag tgcaagtccg gtgcaagcgg ctggatggac tcggctactg gtccaactgg
     4561 tctaaccctg cctacaccgt ggtcatggac atcaaggtgc ccatgagagg ccctgagttc
     4621 tggcggatca tcaacggcga caccatgaag aaagaaaaga acgtgaccct gctgtggaag
     4681 cccctgatga agaacgactc cctgtgctcc gtgcagagat acgtgatcaa ccaccacacc
     4741 agctgcaatg gaacttggag cgaggacgtg ggcaaccaca ccaagtttac ctttctgtgg
     4801 accgagcagg cccacaccgt gactgtgctg gccatcaatt ctatcggcgc cagcgtggcc
     4861 aacttcaatc tgaccttcag ctggcccatg agcaaagtga acatcgtgca gagcctgagc
     4921 gcctatcctc tgaacagcag ctgcgtgatc gtgtcctgga ttctgagccc cagcgactac
     4981 aagctgatgt acttcatcat cgagtggaag aacctgaatg aggacggcga gatcaagtgg
     5041 ctgcggatca gctccagcgt gaagaagtac tacatccacg accacttcat ccccatcgag
     5101 aagtaccagt tctctctgta ccccatcttt atggaaggcg tgggcaagcc caagatcatc
     5161 aatagcttca cccaggatga tattgagaag caccagtccg atggcgcgcc gggcggcgac
     5221 aagacccaca cctgcccccc ctgcccagct ccagaactgc tgggagggcc ttccgtgttc
     5281 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc
     5341 gtggtggtgg ccgtgtccca cgaggaccca gaggtgaagt tcaactggta cgtggacggc
     5401 gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg
     5461 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc
     5521 aaagtctcca acaaggccct ggctgcccca atcgaaaaga caatcagcaa ggccaagggc
     5581 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac
     5641 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg
     5701 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac
     5761 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac
     5821 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
     5881 agcctgagcc ccggcggcag cggtcttaat gatattttcg aggctcagaa gattgagtgg
     5941 catgaacacc accaccatca ccactaaggc cggcctgtag tcactggcac aacgcgttac
     6001 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc
     6061 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct
     6121 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6181 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct
     6241 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6301 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     6361 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6421 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6481 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6541 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6601 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     6661 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     6721 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     6781 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     6841 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     6901 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     6961 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     7021 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     7081 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     7141 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     7201 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     7261 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     7321 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     7381 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7441 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7501 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     7561 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7621 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     7681 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7741 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7801 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     7861 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7921 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7981 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     8041 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8101 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     8161 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8221 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8281 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8341 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8401 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8461 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8521 aagggcgaca cggaaatgtt gaatactcat attcttcctt
//
