LOCUS       sequence-303065-        8413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein partial 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 8413)
  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 8413)
  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-NOTCH3(Trunc2)-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8413
                     /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             5755..5799
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5800..5817
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5957..5974)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5963..6074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6192..6212)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6197..6526
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6377..6512
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6439..6458
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6574..6621
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6640..7239
                     /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(6640..6659)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7136..7156
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7369..7490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7406..7425)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7460..7479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7543..8334,8335..8403))
                     /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     8166..8185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8404..8413,1..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 cggccgccgc tcctccttgt
     2761 cttgatggat ctccttgcgc caacggcggc agatgtacac agctgcctag cagagaagcc
     2821 gcctgccttt gtcctcctgg atgggttgga gagagatgcc agctggaaga tccttgccac
     2881 tctggccctt gtgctggcag aggcgtttgt cagagttctg tggtggccgg cacagccaga
     2941 ttcagctgca gatgccctag aggcttcaga ggccccgatt gctctctgcc cgatccttgt
     3001 ctgagcagcc catgtgctca tggcgccaga tgtagcgtgg gccctgatgg cagattcctg
     3061 tgctcttgtc cacctggcta ccagggcaga tcctgcagat ccgatgtgga cgagtgtaga
     3121 gtgggcgagc cttgtagaca cggcggcacc tgtcttaata cccctggcag cttccggtgc
     3181 cagtgtcctg ctggatatac aggccctctg tgcgagaatc ctgccgtgcc ttgtgctcca
     3241 tctccatgca gaaatggcgg cacatgcaga cagagcggcg acctgacata cgactgtgcc
     3301 tgtctgcctg gcttcgaggg ccagaactgc gaagtgaacg tggacgattg ccctggccac
     3361 agatgcctga atggcggaac ttgtgtggac ggcgtgaaca cctacaactg tcagtgccct
     3421 cctgagtgga ccggccagtt ctgtaccgag gatgtggatg aatgccagct ccagcctaac
     3481 gcctgccaca acggcggaac ctgctttaac acactcggcg gccacagctg cgtgtgtgtg
     3541 aatggatgga caggcgagag ctgcagccag aacatcgatg attgtgccac cgccgtgtgc
     3601 ttccacggcg ccacatgtca tgatagagtg gccagcttct actgcgcctg tcctatgggc
     3661 aagaccggcc tgctgtgtca cctggatgat gcctgcgtgt ccaatccatg ccacgaggac
     3721 gccatctgcg acacaaaccc cgtgaatggc agagccatct gcacctgtcc tccaggcttt
     3781 acaggcggag cctgcgacca ggatgtcgat gagtgtagca tcggcgccaa tccttgcgag
     3841 cacctgggca gatgtgtgaa tacccagggc agctttctgt gtcagtgcgg caggggctac
     3901 acaggcccta gatgtgaaac cgacgtgaac gagtgcctga gcggcccctg tagaaatcag
     3961 gccacctgtc tggaccggat cggccagttt acctgcatct gcatggccgg ctttaccggc
     4021 acatactgcg aggtggacat cgacgagtgc cagtctagcc cctgtgttaa tggcggcgtg
     4081 tgcaaggaca gagtgaacgg cttcagctgt acatgcccct ctggcttcag cggctctacc
     4141 tgtcagctgg acgttgacga gtgtgccagc actccttgtc ggaacggcgc taagtgtgtg
     4201 gaccagcctg atggctacga gtgcagatgt gccgagggct ttgagggcac cctgtgcgac
     4261 agaaatgtcg acgactgcag ccccgatcca tgtcaccacg gaagatgcgt ggacggaatc
     4321 gccagcttta gctgtgcttg tgccccaggc tacaccggca caagatgcga atctcaggtg
     4381 gacgaatgca gaagccagcc ttgcaggcat ggcggcaagt gtctggatct ggtggataag
     4441 tacctgtgtc ggtgcccaag cggcaccaca ggcgtgaact gtgaagtcaa cattgacgac
     4501 tgcgcctcta atccctgcac cttcggcgtg tgtcgcgacg gcatcaacag atacgattgc
     4561 gtgtgtcagc ccggcttcac cggacctctg tgcaacgtgg aaatcaatga gtgcgccagc
     4621 tctccctgcg gcgaaggtgg aagttgtgtc gatggcgaga acggctttcg gtgtctgtgc
     4681 ccacctggat ctctgcctcc actgtgtctg ccaccttctc acccttgcgc tcacgagcca
     4741 tgttctcacg gcatctgcta tgatgcccct ggcggcttta gatgcgtgtg cgaacctgga
     4801 tggtccgggc ctagatgttc tcagagcctg gctagggacg cctgcgagtc tcaaccttgc
     4861 agagccggtg gaacctgtag ctctgatggc atgggcttcc actgcacatg cccaccaggt
     4921 gttcagggca gacagtgcga actgctgagc ccctgcacac ctaatccatg cgaacacggt
     4981 ggcagatgcg agtctgctcc tggacaactg cctgtgtgca gctgtcccca aggatggcaa
     5041 ggccccagat gtcaaggcgc gccgggcggc gacaagaccc acacctgccc cccctgccca
     5101 gctccagaac tgctgggagg gccttccgtg ttcctgttcc cccccaagcc caaggacacc
     5161 ctgatgatca gcaggacccc cgaggtgacc tgcgtggtgg tggccgtgtc ccacgaggac
     5221 ccagaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag
     5281 cccagagagg agcagtacaa cagcacctac agggtggtgt ccgtgctgac cgtgctgcac
     5341 caggactggc tgaacggcaa agaatacaag tgcaaagtct ccaacaaggc cctggctgcc
     5401 ccaatcgaaa agacaatcag caaggccaag ggccagccac gggagcccca ggtgtacacc
     5461 ctgcccccca gccgggagga gatgaccaag aaccaggtgt ccctgacctg tctggtgaag
     5521 ggcttctacc ccagcgatat cgccgtggag tgggagagca acggccagcc cgagaacaac
     5581 tacaagacca cccccccagt gctggacagc gacggcagct tcttcctgta cagcaagctg
     5641 accgtggaca agtccaggtg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag
     5701 gccctgcaca accactacac ccagaagtcc ctgagcctga gccccggcgg cagcggtctt
     5761 aatgatattt tcgaggctca gaagattgag tggcatgaac accaccacca tcaccactaa
     5821 ggccggcctg tagtcactgg cacaacgcgt tacccggtaa gccaatcggg tatacacggt
     5881 cgtcatactg cagacagggt tcttctactt tgcaagatag tcttgagtag taaaataaat
     5941 agatagagaa aatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6001 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6061 gaggaaattg catcacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     6121 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6181 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     6241 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     6301 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     6361 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6421 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     6481 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     6541 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
     6601 atcggcatag tataatacga caaggtgagg aactaaacca tgaccgagta caagcccacg
     6661 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6721 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     6781 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6841 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6901 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     6961 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     7021 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     7081 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacatccgc gccccgcaac
     7141 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     7201 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac acgtgctacg agatttcgat
     7261 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     7321 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     7381 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     7441 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     7501 ataccgtcga cctctagcta gagcttggcg taatcatggt cattaccaat gcttaatcag
     7561 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7621 cgtgtagata actacgatac gggagggctt accatctggc cccagcgctg cgatgatacc
     7681 gcgagaacca cgctcaccgg ctccggattt atcagcaata aaccagccag ccggaagggc
     7741 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7801 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccatcgctac
     7861 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7921 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7981 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8041 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8101 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8161 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8221 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8281 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8341 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8401 catattcttc ctt
//
