LOCUS       sequence-303085-        8611 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 8611)
  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 8611)
  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(Trunc3)-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8611
                     /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             5953..5997
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5998..6015
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6155..6172)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6161..6272
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6390..6410)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6395..6724
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6575..6710
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6637..6656
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6772..6819
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6838..7437
                     /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(6838..6857)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7334..7354
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7567..7688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7604..7623)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7658..7677
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7741..8532,8533..8601))
                     /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     8364..8383
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8602..8611,1..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 cggccgccca ggatgttgat
     2761 gaatgtgctg gacctgctcc ttgcggccct cacggcatct gtacaaatct ggccggcagc
     2821 ttcagctgta cctgtcacgg tggctataca ggccccagct gcgaccagga catcaacgac
     2881 tgcgatccca atccttgcct gaacggcgga tcttgtcagg atggcgtggg cagcttttcc
     2941 tgtagctgtc tgcctggctt cgccggacct agatgtgcca gagatgtgga cgagtgcctg
     3001 agcaatcctt gtggccctgg cacctgtaca gaccacgtgg ccagctttac atgcacctgt
     3061 cctccaggct atggcggctt tcactgcgag caagatctgc ccgattgcag ccccagcagc
     3121 tgttttaatg gcggcacatg tgtggacggc gtgaacagct ttagctgcct gtgcagacct
     3181 ggatacacag gcgcccactg tcagcacgaa gccgatcctt gtctgagcag accttgtctg
     3241 catggcggcg tgtgttctgc cgctcatcct ggctttagat gtacctgcct ggaaagcttc
     3301 acaggccctc agtgtcagac cctggtggat tggtgtagca gacagccctg ccagaatggc
     3361 ggaagatgtg tccagacagg cgcctattgt ctgtgccctc ctggatggtc cggcagactg
     3421 tgcgatatca gaagcctgcc ttgtagagaa gccgccgctc agatcggcgt tagactggaa
     3481 caactgtgtc aggctggcgg acagtgcgtg gacgaggata gcagccacta ctgtgtgtgt
     3541 cctgagggca gaaccggcag ccactgtgaa caagaagtgg acccttgtct ggcccagcct
     3601 tgtcaacacg gtggcacatg tagaggctac atgggcggct atatgtgcga gtgtctgccc
     3661 ggctacaacg gcgacaattg cgaggatgac gtggacgaat gcgcctctca accatgtcaa
     3721 catggcggca gctgtatcga cctggtggcc agatacctgt gcagctgtcc acctggaaca
     3781 ctgggagtgc tgtgcgagat caacgaggat gattgtggcc ccggacctcc tctggattct
     3841 ggacccagat gtctgcacaa cggaacctgt gtggatctcg tcggcggctt taggtgcaca
     3901 tgtccaccag gatataccgg cctgagatgc gaggccgaca tcaatgaatg tagaagcgga
     3961 gcctgtcacg ccgctcacac cagagattgt ctgcaagatc ctggcggcgg attcagatgc
     4021 ctgtgtcatg ccggctttag cggccctaga tgccagacag tgctgagccc ttgcgaatct
     4081 caaccgtgcc agcacggcgg ccaatgtaga ccttctcctg gacctggcgg aggcctgacc
     4141 tttacatgtc actgtgccca gccattttgg ggcccaagat gcgaaagagt ggccagaagc
     4201 tgcagagaac tgcagtgtcc tgtgggagtg ccttgccagc aaacacctag aggccccaga
     4261 tgtgcttgtc ctcctggact gagtggcccc tcctgtagat cttttccagg atctccacca
     4321 ggcgccagca atgcctcttg tgctgctgct ccatgcctcc acggtggaag ttgtagacca
     4381 gctcctctgg cacccttctt cagatgtgcc tgtgctcaag gctggaccgg gccaagatgt
     4441 gaagctcctg ctgctgcacc tgaggtgtcc gaagaaccta ggtgtcctag agccgcctgt
     4501 caggctaaaa ggggcgacca gagatgcgac agagagtgca attctcctgg ctgtggctgg
     4561 gatggcggcg attgcagtct ttctgtgggc gatccttgga ggcagtgcga agctctgcag
     4621 tgctggcggc tgttcaacaa cagcagatgc gatcctgcct gcagcagccc agcctgcctg
     4681 tacgataatt tcgattgcca cgctggcggc agagagcgga cctgtaatcc tgtgtacgag
     4741 aagtactgcg ccgaccactt cgccgacggt agatgtgatc agggctgcaa cacagaggaa
     4801 tgcggctggg acggactgga ttgtgcttct gaagttcccg ctctgctggc tagaggcgtg
     4861 ctggttctta cagttctgct gcctcctgag gaactgctga gaagcagcgc cgatttcctg
     4921 cagagactga gcgctatcct gcggaccagc ctgagattca gactggatgc ccatggacag
     4981 gccatggtgt tcccttacca taggccttct ccaggcagcg agcctagagc cagaagagaa
     5041 ctggcccctg aagtgatcgg cagcgtggtc atgctggaaa tcgacaacag actgtgtctg
     5101 cagagccccg agaacgacca ctgtttccct gatgctcaga gcgccgctga ttatctggga
     5161 gccctgtctg ccgtggaacg gctggatttt ccctatcctc tgagagatgt gcggggcgag
     5221 cctctggaac ctcctgaacc ttctgtgccc cttggcgcgc cgggcggcga caagacccac
     5281 acctgccccc cctgcccagc tccagaactg ctgggagggc cttccgtgtt cctgttcccc
     5341 cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg
     5401 gccgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg
     5461 cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc
     5521 gtgctgaccg tgctgcacca ggactggctg aacggcaaag aatacaagtg caaagtctcc
     5581 aacaaggccc tggctgcccc aatcgaaaag acaatcagca aggccaaggg ccagccacgg
     5641 gagccccagg tgtacaccct gccccccagc cgggaggaga tgaccaagaa ccaggtgtcc
     5701 ctgacctgtc tggtgaaggg cttctacccc agcgatatcg ccgtggagtg ggagagcaac
     5761 ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc
     5821 ttcctgtaca gcaagctgac cgtggacaag tccaggtggc agcagggcaa cgtgttcagc
     5881 tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gagcctgagc
     5941 cccggcggca gcggtcttaa tgatattttc gaggctcaga agattgagtg gcatgaacac
     6001 caccaccatc accactaagg ccggcctgta gtcactggca caacgcgtta cccggtaagc
     6061 caatcgggta tacacggtcg tcatactgca gacagggttc ttctactttg caagatagtc
     6121 ttgagtagta aaataaatag atagagaaaa tcagcctcga ctgtgccttc tagttgccag
     6181 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     6241 gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc tcggtctatt
     6301 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6361 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     6421 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     6481 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     6541 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     6601 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     6661 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     6721 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     6781 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     6841 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     6901 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     6961 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     7021 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     7081 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     7141 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     7201 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     7261 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     7321 acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     7381 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacac
     7441 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     7501 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     7561 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     7621 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     7681 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     7741 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7801 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7861 cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa
     7921 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7981 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     8041 cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8101 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8161 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8221 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     8281 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     8341 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8401 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8461 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     8521 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8581 acggaaatgt tgaatactca tattcttcct t
//
