LOCUS       sequence-481684-        8527 bp    DNA     circular SYN 01-AUG-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 8527)
  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 8527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 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-PTGFRN-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8527
                     /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             5869..5913
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5914..5931
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6071..6088)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6077..6188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6306..6326)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6311..6640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6491..6626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6553..6572
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6688..6735
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6754..7353
                     /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(6754..6773)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7250..7270
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7483..7604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7520..7539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7574..7593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7657..8448,8449..8517))
                     /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     8280..8299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8518..8527,1..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 tcctacagcc
     2761 acacttgtta gagtcgtggg aaccgagctg gtcatcccct gcaacgtgtc cgattacgat
     2821 ggccccagcg agcagaactt cgactggtct tttagcagcc tgggcagcag ctttgtggaa
     2881 ctggcctcta cctgggaagt gggctttccc gctcagctgt accaagagag actgcagaga
     2941 ggcgagatcc tgctgagaag aaccgccaac gatgccgtgg aactgcacat caagaacgtg
     3001 cagcctagcg accagggcca ctacaagtgt agcacaccta gcaccgatgc caccgtgcag
     3061 ggcaattacg aggacaccgt gcaagtgaag gtgctggccg attctctgca cgtgggacca
     3121 tctgctagac ctccacctag cctgtctctg agagagggcg agcctttcga gctgagatgt
     3181 acagccgctt ctgcctctcc tctgcacaca catctggctc tgctgtggga agtgcataga
     3241 ggcccagcta gaagatccgt gctggccctg acacacgagg gcagatttca ccctggactg
     3301 ggctacgagc agagatacca tagcggagat gtgcggctgg ataccgtggg ctctgatgcc
     3361 tacagactgt ctgtgtcaag agccctgagc gccgatcagg gcagctacag atgtatcgtg
     3421 tctgagtgga tcgccgagca aggcaactgg caagagatcc aagagaaggc cgtggaagtg
     3481 gccacagtgg tcattcagcc atctgtgctg agagccgccg tgcctaaaaa tgtgtctgtg
     3541 gccgagggca aagagctgga cctgacctgc aacatcacca ccgacagagc cgatgatgtc
     3601 cggcctgaag tgacctggtc cttctccaga atgcccgata gcacactgcc tggaagcaga
     3661 gtgctggcca gactggatag agatagcctg gtgcactcta gccctcacgt ggccctgtct
     3721 catgtggacg ccagatccta tcatctgctt gtgcgggacg tgtccaaaga gaacagcggc
     3781 tactactact gccacgtgtc actgtgggcc cctggccaca atagatcttg gcacaaagtg
     3841 gccgaagccg tgtcatctcc tgctggcgtt ggagtgacat ggctggaacc cgattaccag
     3901 gtgtacctga atgccagcaa ggtgcccggc tttgccgatg atcctacaga gctggcctgc
     3961 agagtggtgg atacaaagtc tggcgaggcc aacgtgcggt tcaccgtgtc ctggtactac
     4021 agaatgaacc ggcggagcga caacgtggtc acatctgaac tgctggccgt gatggatggc
     4081 gactggacac tgaagtacgg cgagagatct aagcagagag cccaggacgg cgacttcatc
     4141 ttcagcaaag agcacaccga caccttcaac ttcagaatcc agcggaccac cgaagaggac
     4201 cggggcaatt attactgtgt ggtgtccgcc tggaccaagc agagaaacaa cagctgggtc
     4261 aagagcaagg acgtgttcag caagcccgtg aacatcttct gggccctcga ggattctgtg
     4321 ctggtggtta aggccagaca gcccaagcct ttctttgccg ccggaaacac cttcgagatg
     4381 acatgcaagg tgtccagcaa gaacatcaag tccccacggt acagcgtgct gatcatggcc
     4441 gagaaacctg tgggcgacct gagcagcccc aacgagacaa agtacatcat ctccctggac
     4501 caggacagcg tggtcaagct ggaaaactgg accgacgctt ccagagtgga tggcgtggtg
     4561 ctggaaaagg tgcaagagga cgagttccgg tacaggatgt accagacaca ggtgtccgat
     4621 gccggcctgt acagatgcat ggttacagct tggagccctg tgcggggaag cctttggaga
     4681 gaagctgcca caagcctgag caaccctatc gagatcgact tccagacaag cggccccatc
     4741 ttcaatgcca gcgtgcactc tgatacccct agcgtgatcc ggggcgatct gatcaagctg
     4801 ttctgcatca tcaccgtgga aggcgccgct ctggaccctg atgatatggc cttcgatgtg
     4861 tcttggtttg ccgtgcacag cttcggcctg gataaggctc ctgttctgct gtcctctctg
     4921 gacagaaagg gcatcgtgac cacctccaga agagactgga agtctgacct gagcctggaa
     4981 agggtgtccg tgctcgagtt tctgctgcaa gtgcacggct ccgaggacca ggatttcggc
     5041 aactactatt gcagcgtgac cccttgggtc aagtctccta caggcagctg gcagaaagag
     5101 gccgagattc acagcaagcc tgtgtttatc accgtgaaga tggacgtgct gaacgccttc
     5161 aagtaccctg gcgcgccggg cggcgacaag acccacacct gccccccctg cccagctcca
     5221 gaactgctgg gagggccttc cgtgttcctg ttccccccca agcccaagga caccctgatg
     5281 atcagcagga cccccgaggt gacctgcgtg gtggtggccg tgtcccacga ggacccagag
     5341 gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccaga
     5401 gaggagcagt acaacagcac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac
     5461 tggctgaacg gcaaagaata caagtgcaaa gtctccaaca aggccctggc tgccccaatc
     5521 gaaaagacaa tcagcaaggc caagggccag ccacgggagc cccaggtgta caccctgccc
     5581 cccagccggg aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc
     5641 taccccagcg atatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag
     5701 accacccccc cagtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg
     5761 gacaagtcca ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg
     5821 cacaaccact acacccagaa gtccctgagc ctgagccccg gcggcagcgg tcttaatgat
     5881 attttcgagg ctcagaagat tgagtggcat gaacaccacc accatcacca ctaaggccgg
     5941 cctgtagtca ctggcacaac gcgttacccg gtaagccaat cgggtataca cggtcgtcat
     6001 actgcagaca gggttcttct actttgcaag atagtcttga gtagtaaaat aaatagatag
     6061 agaaaatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6121 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     6181 attgcatcac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     6241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     6301 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     6361 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     6421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     6481 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     6541 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     6601 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     6661 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     6721 atagtataat acgacaaggt gaggaactaa accatgaccg agtacaagcc cacggtgcgc
     6781 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     6841 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     6901 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     6961 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     7021 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     7081 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     7141 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     7201 gcggccgagc gcgccggggt gcccgccttc ctggagacat ccgcgccccg caacctcccc
     7261 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     7321 acctggtgca tgacccgcaa gcccggtgcc tgacacgtgc tacgagattt cgattccacc
     7381 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     7441 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     7501 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     7561 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     7621 tcgacctcta gctagagctt ggcgtaatca tggtcattac caatgcttaa tcagtgaggc
     7681 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7741 gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga
     7801 accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg
     7861 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7921 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat
     7981 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8041 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8101 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8161 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8221 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8281 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8341 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8401 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8461 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt
     8521 cttcctt
//
