LOCUS       sequence-303113-        8827 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 8827)
  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 8827)
  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-NOTCH1(Trunc3)-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8827
                     /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             6169..6213
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6214..6231
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6371..6388)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6377..6488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6606..6626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6611..6940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6791..6926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6853..6872
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6988..7035
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7054..7653
                     /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(7054..7073)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7550..7570
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7783..7904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7820..7839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7874..7893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7957..8748,8749..8817))
                     /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     8580..8599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8818..8827,1..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 cggccgccgt tcttgctcct
     2761 tgtgctccat ctccttgcag aaacggcggc gagtgtagac agagcgagga ctacgagagc
     2821 ttcagctgtg tgtgtccaac aggctggcag ggccagacct gcgaggtgga catcaatgag
     2881 tgtgtgctga gcccttgtag acacggcgcc agctgtcaga atacccacgg cggctataga
     2941 tgtcactgtc aggccggcta cagcggcaga aactgcgaga cagacatcga cgactgcaga
     3001 cccaatcctt gccacaatgg cggcagctgc accgacggaa tcaataccgc cttctgcgat
     3061 tgcctgcctg gcttcagagg caccttctgt gaagaggata tcaacgagtg cgccagcgat
     3121 ccctgcagaa atggcgccaa ttgcaccgac tgcgtggaca gctacacctg tacatgtcct
     3181 gccggcttta gcggcatcca ctgcgagaac aatacccctg actgcaccga gtccagctgc
     3241 tttaatggcg gaacctgtgt ggacggcatc aattccttca cctgtctgtg cccacctggc
     3301 tttaccggca gctactgcca gcacgatgtg aatgagtgcg acagccagcc ttgtctgcac
     3361 ggcggaactt gtcaggatgg ctgcggctcc tacagatgta cctgtcctca gggctacaca
     3421 ggccccaact gccagaatct ggtgcattgg tgcgatagca gcccctgcaa gaatggcggg
     3481 aagtgttggc agacccacac acagtacaga tgcgagtgtc cttctggctg gaccggcctg
     3541 tattgtgatg tgccttctgt gtcctgcgaa gtggccgctc aaagacaggg tgttgatgtg
     3601 gccagactgt gtcagcatgg cggcctgtgt gttgatgccg gcaataccca tcactgtcgg
     3661 tgccaggccg gatacacagg ctcttattgc gaggacctgg tggacgagtg tagcccaagt
     3721 ccttgtcaga atggggccac ctgtaccgat tacctcggcg gctactcctg taaatgcgtg
     3781 gccggatatc acggcgtgaa ctgcagcgaa gagatcgacg agtgcctgtc tcacccctgt
     3841 caaaacggcg ggacatgcct ggacctgcct aacacctaca agtgcagctg tcctagagga
     3901 acccagggcg tgcactgcga aatcaacgtg gacgattgca accctcctgt ggaccccgtg
     3961 tccagatctc ccaagtgctt caacaacggc acatgcgtgg accaagtcgg cggatactct
     4021 tgcacatgcc ctccaggctt tgtgggcgag agatgtgaag gcgacgtgaa cgagtgtctg
     4081 agcaaccctt gtgacgccag aggcacacag aactgcgtgc agagagtgaa cgactttcac
     4141 tgcgagtgca gagccggcca cactgggaga agatgcgaga gcgtgatcaa cggctgcaag
     4201 ggcaagccct gcaaaaacgg tggaacatgc gccgtggcct ctaataccgc caggggcttt
     4261 atctgcaagt gccctgctgg atttgagggc gccacatgcg agaatgatgc cagaacatgc
     4321 ggcagcctga gatgccttaa cggcggcacc tgtatcagcg gccctagaag ccctacatgt
     4381 ctgtgtctgg gccctttcac aggccctgag tgccagtttc ctgccagctc tccttgtctc
     4441 ggcggcaacc catgttacaa ccagggcacc tgtgaaccca ccagcgagag ccccttttac
     4501 agatgcctgt gtccagccaa gttcaacggc ctgctgtgcc acatcctgga ttatagcttt
     4561 ggcggcggag ccggcagaga tatccctcca cctctgattg aggaagcctg cgagctgccc
     4621 gagtgtcaag aggatgctgg caacaaagtg tgcagcctgc agtgcaacaa tcacgcctgt
     4681 ggatgggatg gcggcgactg cagcctgaac ttcaacgacc cttggaagaa ctgcacccag
     4741 tctctgcagt gctggaagta ctttagcgac ggccactgcg actcccagtg caattctgcc
     4801 ggatgcctgt tcgacggctt cgattgtcag cgagccgagg gccagtgtaa ccctctgtac
     4861 gaccagtact gcaaggacca cttctccgat ggccactgtg accagggatg taatagcgcc
     4921 gagtgcgagt gggatggact ggattgtgcc gagcacgtgc cagaaagact ggctgctgga
     4981 acactggtgg tggtcgtgct gatgcctcct gagcagctga gaaacagcag cttccacttc
     5041 ctgcgcgagc tgagcagagt gctgcacacc aatgtggtgt tcaagagaga tgcccacggc
     5101 cagcagatga tcttcccata ttacggcaga gaagaggaac tgcggaagca ccctatcaag
     5161 agagctgctg aaggctgggc tgcccctgat gctctgcttg gacaagtgaa agcttctctg
     5221 ctgcctggcg gaagcgaagg cggaaggcgt agaagagaac tggaccctat ggatgtgcgg
     5281 ggcagcatcg tgtacctgga aatcgacaac agacagtgcg tgcaggcctc cagccagtgt
     5341 tttcagtctg caaccgatgt ggccgccttt ctgggagcac tggcttctct gggaagcctg
     5401 aacatcccct acaagatcga ggccgtgcag tccgagacag tggaacctcc tcctcctgct
     5461 caactgcatg gcgcgccggg cggcgacaag acccacacct gccccccctg cccagctcca
     5521 gaactgctgg gagggccttc cgtgttcctg ttccccccca agcccaagga caccctgatg
     5581 atcagcagga cccccgaggt gacctgcgtg gtggtggccg tgtcccacga ggacccagag
     5641 gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccaga
     5701 gaggagcagt acaacagcac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac
     5761 tggctgaacg gcaaagaata caagtgcaaa gtctccaaca aggccctggc tgccccaatc
     5821 gaaaagacaa tcagcaaggc caagggccag ccacgggagc cccaggtgta caccctgccc
     5881 cccagccggg aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc
     5941 taccccagcg atatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag
     6001 accacccccc cagtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg
     6061 gacaagtcca ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg
     6121 cacaaccact acacccagaa gtccctgagc ctgagccccg gcggcagcgg tcttaatgat
     6181 attttcgagg ctcagaagat tgagtggcat gaacaccacc accatcacca ctaaggccgg
     6241 cctgtagtca ctggcacaac gcgttacccg gtaagccaat cgggtataca cggtcgtcat
     6301 actgcagaca gggttcttct actttgcaag atagtcttga gtagtaaaat aaatagatag
     6361 agaaaatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6421 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     6481 attgcatcac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     6541 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     6601 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     6661 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     6721 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     6781 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     6841 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     6901 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     6961 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     7021 atagtataat acgacaaggt gaggaactaa accatgaccg agtacaagcc cacggtgcgc
     7081 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     7141 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     7201 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     7261 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     7321 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     7381 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     7441 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     7501 gcggccgagc gcgccggggt gcccgccttc ctggagacat ccgcgccccg caacctcccc
     7561 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     7621 acctggtgca tgacccgcaa gcccggtgcc tgacacgtgc tacgagattt cgattccacc
     7681 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     7741 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     7801 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     7861 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     7921 tcgacctcta gctagagctt ggcgtaatca tggtcattac caatgcttaa tcagtgaggc
     7981 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     8041 gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga
     8101 accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg
     8161 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     8221 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat
     8281 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8341 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8401 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8461 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8521 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8581 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8641 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8701 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8761 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt
     8821 cttcctt
//
