LOCUS       sequence-302687-        7147 bp    DNA     circular SYN 15-DEC-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 7147)
  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 7147)
  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-UNC5D-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7147
                     /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             4489..4533
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4534..4551
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4691..4708)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4697..4808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4926..4946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4931..5260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5111..5246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5173..5192
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5308..5355
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5374..5973
                     /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(5374..5393)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5870..5890
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6103..6224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6140..6159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6194..6213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6277..7068,7069..7137))
                     /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     6900..6919
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7138..7147,1..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 aagaggaaca
     2761 gataatggcg aagccctgcc tgagagcatc ccatctgcac ctggaacact gccccacttc
     2821 atcgaggaac ccgacgacgc ctacatcatc aagagcaacc ctatcgctct gcggtgcaag
     2881 gccagacctg ccatgcagat ctttttcaag tgcaacggcg agtgggtgca ccagaacgag
     2941 cacgtgtccg aggaaacact ggacgagagc agcggcctga aagtgcgcga ggtgttcatc
     3001 aacgtgaccc ggcagcaggt cgaggacttt cacggccctg aggattactg gtgccagtgt
     3061 gtggcttgga gccacctggg cacaagcaag agcagaaaag cctctgtgcg gatcgcctac
     3121 ctgcggaaga acttcgagca ggaccctcag ggcagagagg tgccaatcga gggaatgatc
     3181 gtgctgcact gcagacctcc agaaggcgtg ccagctgctg aagtggaatg gctgaagaac
     3241 gaggaaccta tcgacagcga gcaggatgag aacatcgaca ccagagccga ccacaacctg
     3301 atcatcagac aggccagact gagcgacagc ggcaactaca cttgcatggc cgccaacatc
     3361 gtggccaaga gaagatctct gagcgccacc gtggtggtgt atgtgaatgg cggatggtcc
     3421 tcctggacag agtggtccgc ctgcaacgtc cgatgtggaa gaggctggca gaagcggagc
     3481 agaacctgta caaaccccgc tcctcttaac ggcggagcct tttgcgaagg catgagcgtg
     3541 cagaagatta cctgtacctc tctgtgcccc gtggacggct cttgggaagt ttggagcgag
     3601 tggtctgtgt gcagccccga gtgtgaacac ctgagaatca gagagtgcac agcccctcca
     3661 cctagaaacg gcggcaagtt ttgcgagggc ctgtctcaag agagcgagaa ctgtaccgac
     3721 ggcctgtgca tcctggacaa gaagcctctg cacgagatca agccccagag catcgagaac
     3781 gcctccgatg gcgcgccggg cggcgacaag acccacacct gccccccctg cccagctcca
     3841 gaactgctgg gagggccttc cgtgttcctg ttccccccca agcccaagga caccctgatg
     3901 atcagcagga cccccgaggt gacctgcgtg gtggtggccg tgtcccacga ggacccagag
     3961 gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccaga
     4021 gaggagcagt acaacagcac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac
     4081 tggctgaacg gcaaagaata caagtgcaaa gtctccaaca aggccctggc tgccccaatc
     4141 gaaaagacaa tcagcaaggc caagggccag ccacgggagc cccaggtgta caccctgccc
     4201 cccagccggg aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc
     4261 taccccagcg atatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag
     4321 accacccccc cagtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg
     4381 gacaagtcca ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg
     4441 cacaaccact acacccagaa gtccctgagc ctgagccccg gcggcagcgg tcttaatgat
     4501 attttcgagg ctcagaagat tgagtggcat gaacaccacc accatcacca ctaaggccgg
     4561 cctgtagtca ctggcacaac gcgttacccg gtaagccaat cgggtataca cggtcgtcat
     4621 actgcagaca gggttcttct actttgcaag atagtcttga gtagtaaaat aaatagatag
     4681 agaaaatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4741 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4801 attgcatcac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4861 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     4921 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4981 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     5041 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     5101 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     5161 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     5221 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     5281 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     5341 atagtataat acgacaaggt gaggaactaa accatgaccg agtacaagcc cacggtgcgc
     5401 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     5461 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     5521 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     5581 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     5641 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5701 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     5761 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5821 gcggccgagc gcgccggggt gcccgccttc ctggagacat ccgcgccccg caacctcccc
     5881 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5941 acctggtgca tgacccgcaa gcccggtgcc tgacacgtgc tacgagattt cgattccacc
     6001 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     6061 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     6121 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6181 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     6241 tcgacctcta gctagagctt ggcgtaatca tggtcattac caatgcttaa tcagtgaggc
     6301 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6361 gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga
     6421 accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg
     6481 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6541 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat
     6601 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6661 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6721 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6781 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6841 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6901 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6961 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7021 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7081 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt
     7141 cttcctt
//
