LOCUS       sequence-303093-        8644 bp    DNA     circular SYN 15-APR-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 8644)
  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 8644)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-NOTCH2(Trunc3)-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8644
                     /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             5986..6030
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6031..6048
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6188..6205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6194..6305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6423..6443)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6428..6757
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6608..6743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6670..6689
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6805..6852
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6871..7470
                     /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(6871..6890)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7367..7387
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7600..7721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7637..7656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7691..7710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7774..8565,8566..8634))
                     /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     8397..8416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8635..8644,1..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 cggccgccac agttcttgct
     2761 ccatgcagcc ctaatccttg cgagaatgcc gccgtgtgca aagagagccc caacttcgag
     2821 agctacacct gtctgtgtgc ccctggctgg cagggccaga gatgtaccat cgacatcgac
     2881 gagtgcatca gcaagccctg catgaaccac ggcctgtgcc acaataccca gggcagctac
     2941 atgtgcgagt gccctccagg ctttagcggc atggactgcg aagaggacat cgatgactgc
     3001 ctggccaatc catgccagaa tggcggcagc tgtatggacg gcgtgaacac ctttagctgc
     3061 ctgtgcctgc ctggcttcac cggcgataag tgccagaccg acatgaacga gtgcctgagc
     3121 gagccttgca agaacggcgg cacatgcagc gactacgtga acagctacac atgcaagtgc
     3181 caggccggct tcgatggcgt gcactgcgag aacaacatca atgagtgcac cgagtccagc
     3241 tgcttcaacg gcggaacctg tgtggacggc atcaatagct tctcttgtct gtgccccgtg
     3301 ggctttaccg gcagcttttg tctgcacgag atcaacgagt gcagctctca cccctgcctg
     3361 aacgagggca catgtgtgga tggcctgggc acctacagat gctcttgtcc tctgggctac
     3421 accggcaaga attgtcagac cctggtcaac ctgtgcagca gatccccatg caagaacaag
     3481 ggaacctgcg tgcagaagaa agccgagagc cagtgcctgt gtccttctgg atgggctggc
     3541 gcctactgtg atgtgcccaa tgtgtcctgt gatatcgccg ccagtagacg gggagtgctg
     3601 gtggaacatc tgtgtcagca cagcggcgtg tgcatcaacg ccggcaatac ccactactgc
     3661 cagtgtccac tgggatacac aggctcctac tgcgaggaac agctggacga gtgtgccagc
     3721 aatccttgtc agcacggcgc cacctgtagc gatttcatcg gcggctatag atgcgagtgc
     3781 gtgccaggat atcagggcgt gaactgtgaa tacgaggtgg acgaatgcca gaaccagcct
     3841 tgtcaaaatg gcgggacctg catcgacctc gtgaaccact tcaagtgcag ctgccctcct
     3901 ggcacaagag gcctgctgtg cgaggaaaac attgacgact gcgccagagg accccactgt
     3961 ctgaatggtg gccagtgcat ggatcggatc ggaggctaca gctgcagatg tctgcctgga
     4021 tttgccggcg agagatgcga gggcgacatc aacgaatgtc tgtctaaccc ctgcagcagc
     4081 gagggcagcc tggattgcat ccagctgacc aacgactacc tgtgcgtgtg cagaagcgcc
     4141 ttcactggca gacactgcga aaccttcgtg gacgtgtgcc ctcagatgcc ctgtctcaat
     4201 ggcggaacat gcgccgtggc cagcaatatg cctgacggct tcatctgccg gtgtcctcca
     4261 ggattttcag gcgccagatg ccagagcagc tgcggccaag tgaagtgcag aaagggcgag
     4321 cagtgcgtgc acacagccag cggacctaga tgcttctgcc catctcctag agactgcgag
     4381 agcggctgtg cttctagccc ttgccaacac ggcggaagct gccatcctca gagacagcca
     4441 ccttactaca gctgtcagtg cgccccacct ttcagcggaa gcagatgcga gctgtacaca
     4501 gcccctccaa gcacacctcc tgccacatgt ctgagccagt actgcgccga caaggctaga
     4561 gatggcgtgt gtgacgaggc ctgcaatagc catgcctgtc agtgggatgg cggcgactgt
     4621 agcctgacca tggaaaaccc ttgggccaac tgcagctccc ctctgccttg ctgggactac
     4681 atcaacaacc agtgcgacga gctgtgcaac accgtggaat gcctgttcga caatttcgag
     4741 tgccagggca actccaagac ctgcaaatac gacaagtact gtgccgatca cttcaaggac
     4801 aaccactgcg atcagggctg caacagcgaa gagtgtggct gggacggact ggattgtgct
     4861 gccgaccagc cagagaatct ggccgaggga acactggtca tcgtggtgct gatgccacct
     4921 gaacagctgc tccaggacgc cagaagcttt ctgagagccc tgggcacact gctgcatacc
     4981 aacctgcgga tcaagaggga tagccagggc gagctgatgg tgtaccccta ctacggcgaa
     5041 aagtccgccg ccatgaagaa acagagaatg accaggcgca gcctgccagg cgagcaagag
     5101 caagaagtgg ccggctctaa ggtgttcctg gaaatcgaca accggcagtg tgtgcaggac
     5161 agcgaccact gcttcaagaa caccgatgct gccgctgctc tgctggcctc tcatgccatt
     5221 cagggaaccc tgagctaccc tctggtgtcc gtggtgtctg agtctctgac ccctgagaga
     5281 acccaaggcg cgccgggcgg cgacaagacc cacacctgcc ccccctgccc agctccagaa
     5341 ctgctgggag ggccttccgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc
     5401 agcaggaccc ccgaggtgac ctgcgtggtg gtggccgtgt cccacgagga cccagaggtg
     5461 aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagagag
     5521 gagcagtaca acagcaccta cagggtggtg tccgtgctga ccgtgctgca ccaggactgg
     5581 ctgaacggca aagaatacaa gtgcaaagtc tccaacaagg ccctggctgc cccaatcgaa
     5641 aagacaatca gcaaggccaa gggccagcca cgggagcccc aggtgtacac cctgcccccc
     5701 agccgggagg agatgaccaa gaaccaggtg tccctgacct gtctggtgaa gggcttctac
     5761 cccagcgata tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc
     5821 acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac
     5881 aagtccaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac
     5941 aaccactaca cccagaagtc cctgagcctg agccccggcg gcagcggtct taatgatatt
     6001 ttcgaggctc agaagattga gtggcatgaa caccaccacc atcaccacta aggccggcct
     6061 gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg tcgtcatact
     6121 gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa tagatagaga
     6181 aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     6241 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6301 gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6361 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     6421 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     6481 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     6541 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     6601 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     6661 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     6721 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     6781 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     6841 gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac ggtgcgcctc
     6901 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6961 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     7021 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     7081 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     7141 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     7201 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     7261 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     7321 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     7381 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     7441 tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga ttccaccgcc
     7501 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     7561 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     7621 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     7681 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     7741 acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca gtgaggcacc
     7801 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7861 aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc
     7921 acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7981 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     8041 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt
     8101 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     8161 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     8221 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     8281 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8341 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8401 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8461 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8521 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8581 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt
     8641 cctt
//
