LOCUS       sequence-489479-        8194 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8194)
  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 8194)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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-SIGLEC8-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8194
                     /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"
     regulatory      2743..2752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3844..3867
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5527..5571
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5572..5598
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5738..5755)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5744..5855
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5973..5993)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5978..6307
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6158..6293
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6220..6239
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6355..6402
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6421..7020
                     /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(6421..6440)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6917..6937
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7150..7271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7187..7206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7241..7260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7324..8115,8116..8184))
                     /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     7947..7966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8185..8194,1..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 cggccgccat ggaaggcgat
     2761 agacaatacg gcgacggcta cctgctgcag gtccaagagc tggtcacagt gcaagagggc
     2821 ctgtgtgtgc acgtgccctg cagctttagc taccctcagg atggctggac cgacagcgat
     2881 cctgtgcacg gctattggtt cagagccggc gacagaccct atcaggatgc ccctgtggcc
     2941 accaacaatc ccgatagaga ggtgcaggcc gagacacagg gcagatttca gctgctgggc
     3001 gacatctggt ccaacgactg tagcctgagc atccgggacg cccggaagag agataagggc
     3061 agctacttct tccggctgga acggggcagc atgaagtggt cctacaagag ccagctgaac
     3121 tacaagacca agcagctgag cgtgttcgtg acagccctga cacacagacc cgacatcctg
     3181 atcctgggca cactggaatc tggccacagc agaaacctga cctgtagcgt gccctgggcc
     3241 tgcaaacagg gaacccctcc tatgatctct tggatcggcg ccagcgtgtc ctctcctgga
     3301 cctacaacag ctagaagcag cgtgctgacc ctgacaccta agcctcagga tcacggcacc
     3361 agcctgacct gccaagttac acttcctggc accggcgtga ccaccacaag cacagttaga
     3421 ctggacgtgt cctatcctcc ttggaacctg accatgaccg tgttccaggg cgacgccaca
     3481 gcctctacag ccctcggaaa tggcagcagc ctgtctgtgc tggaaggcca gtctctgaga
     3541 ctcgtgtgcg ccgtgaacag caaccctcct gctagactga gctggaccag aggcagcctg
     3601 acactgtgtc ctagcagaag cagcaatccc ggcctgctgg aactgcctag agtgcacgtt
     3661 cgagatgagg gcgagttcac ctgtagagcc cagaatgccc agggctccca gcacatctct
     3721 ctgagcctga gcctgcagaa tgaaggcacc ggaaccagca gacccgtgtc tcaagtgaca
     3781 ctggctgctg gcgcgccgga agatcaagtt gatccaagat tgattgatgg caagggcgga
     3841 tctctggagg tgctgttcca gggaccaggc ggaagcggca atcccggcgg cgatctggcc
     3901 ccccagatgc tgcgcgagct gcaggagacc aatgccgccc tgcaggacgt gcgcgagctg
     3961 ctgcgccacc gcgtgaagga gatcaccttc ctgaagaaca ccgtgatgga gtgcgacgcc
     4021 tgcggtatgc agccctctag aggctccggc tccatcatcc cagttgagga ggagaacccg
     4081 gacttctgga accgcgaggc agccgaggcc ctgggtgccg ccaagaagct gcagcctgca
     4141 cagacagccg ccaagaacct catcatcttc ctgggcgatg ggatgggggt gtctacggtg
     4201 acagctgcca ggatcctaaa agggcagaag aaggacaaac tggggcctga gatacccctg
     4261 gccatggacc gcttcccata tgtggctctg tccaagacat acaatgtaga caaacatgtg
     4321 ccagacagtg gagccacagc cacggcctac ctgtgcgggg tcaagggcaa cttccagacc
     4381 attggcttga gtgcagccgc ccgctttaac cagtgcaaca cgacacgcgg caacgaggtc
     4441 atctccgtga tgaatcgggc caagaaagca gggaagtcag tgggagtggt aaccaccaca
     4501 cgagtgcagc acgcctcgcc agccggcacc tacgcccaca cggtgaaccg caactggtac
     4561 tcggacgccg acgtgcctgc ctcggcccgc caggaggggt gccaggacat cgctacgcag
     4621 ctcatctcca acatggacat tgacgtgatc ctaggtggag gccgaaagta catgtttcgc
     4681 atgggaaccc cagaccctga gtacccagat gactacagcc aaggtgggac caggctggac
     4741 gggaagaatc tggtgcagga atggctggcg aagcgccagg gtgcccggta tgtgtggaac
     4801 cgcactgagc tcatgcaggc ttccctggac ccgtctgtga cccatctcat gggtctcttt
     4861 gagcctggag acatgaaata cgagatccac cgagactcca cactggaccc ctccctgatg
     4921 gagatgacag aggctgccct gcgcctgctg agcaggaacc cccgcggctt cttcctcttc
     4981 gtggagggtg gtcgcatcga ccatggtcat catgaaagca gggcttaccg ggcactgact
     5041 gagacgatca tgttcgacga cgccattgag agggcgggcc agctcaccag cgaggaggac
     5101 acgctgagcc tcgtcactgc cgaccactcc cacgtcttct ccttcggagg ctaccccctg
     5161 cgagggagct ccatcttcgg gctggcccct ggcaaggccc gggacaggaa ggcctacacg
     5221 gtcctcctat acggaaacgg tccaggctat gtgctcaagg acggcgcccg gccggatgtt
     5281 accgagagcg agagcgggag ccccgagtat cggcagcagt cagcagtgcc cctggacgaa
     5341 gagacccacg caggcgagga cgtggcggtg ttcgcgcgcg gcccgcaggc gcacctggtt
     5401 cacggcgtgc aggagcagac cttcatagcg cacgtcatgg ccttcgccgc ctgcctggag
     5461 ccctacaccg cctgcgacct ggcgcccccc gccggcacca ccgacgccgc gcacccgggt
     5521 ggcggaggac tgaacgacat cttcgaggct cagaaaatcg aatggcacga acatcaccat
     5581 caccatcacc atcaccatta aggccggcct gtagtcactg gcacaacgcg ttacccggta
     5641 agccaatcgg gtatacacgg tcgtcatact gcagacaggg ttcttctact ttgcaagata
     5701 gtcttgagta gtaaaataaa tagatagaga aaatcagcct cgactgtgcc ttctagttgc
     5761 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5821 actgtccttt cctaataaaa tgaggaaatt gcatcacaac actcaaccct atctcggtct
     5881 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5941 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     6001 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6061 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     6121 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     6181 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     6241 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     6301 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     6361 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     6421 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     6481 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     6541 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     6601 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     6661 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     6721 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     6781 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     6841 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     6901 gagacatccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     6961 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     7021 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     7081 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     7141 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     7201 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     7261 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     7321 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7381 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7441 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     7501 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7561 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7621 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7681 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7741 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7801 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7861 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7921 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7981 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8041 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8101 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8161 gacacggaaa tgttgaatac tcatattctt cctt
//
