LOCUS       sequence-303609-        7858 bp    DNA     circular SYN 15-APR-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 7858)
  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 7858)
  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-TNFRSF1B-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7858
                     /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             3508..3531
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5191..5235
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5236..5262
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5402..5419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5408..5519
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5637..5657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5642..5971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5822..5957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5884..5903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6019..6066
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6085..6684
                     /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(6085..6104)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6581..6601
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6814..6935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6851..6870)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6905..6924
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6988..7779,7780..7848))
                     /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     7611..7630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7849..7858,1..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 cggccgcctt gcctgctcaa
     2761 gtggccttta caccttatgc tcccgagcct ggcagcacct gtagactgag agagtactac
     2821 gaccagacag cccagatgtg ctgctccaag tgttctcctg gacagcacgc caaggtgttc
     2881 tgcaccaaga ccagcgatac cgtgtgcgac agctgcgagg atagcacata cacccagctg
     2941 tggaattggg tgcccgagtg tctgagctgt ggcagcagat gtagcagcga ccaggtggaa
     3001 acccaggcct gcaccagaga gcagaaccgg atctgtacat gcagacctgg ctggtactgc
     3061 gccctgtcta agcaagaggg atgcagactg tgcgcccctc tgagaaagtg tagacccggc
     3121 tttggagtgg ccagaccagg cacagagaca agcgacgttg tgtgcaagcc ttgcgctccc
     3181 ggcaccttta gcaataccac cagcagcacc gacatctgca gaccccacca gatctgcaac
     3241 gtggtggcca ttcctggcaa tgccagcatg gatgccgtgt gcacatctac aagccctacc
     3301 agatctatgg cccctggcgc tgttcatctg cctcagcctg tgtctaccag aagccagcac
     3361 acacagccta cacctgagcc tagcacagcc cctagcacaa gctttctgct gcccatggga
     3421 ccttctccac cagccgaagg atctaccggt gatggcgcgc cggaagatca agttgatcca
     3481 agattgattg atggcaaggg cggatctctg gaggtgctgt tccagggacc aggcggaagc
     3541 ggcaatcccg gcggcgatct ggccccccag atgctgcgcg agctgcagga gaccaatgcc
     3601 gccctgcagg acgtgcgcga gctgctgcgc caccgcgtga aggagatcac cttcctgaag
     3661 aacaccgtga tggagtgcga cgcctgcggt atgcagccct ctagaggctc cggctccatc
     3721 atcccagttg aggaggagaa cccggacttc tggaaccgcg aggcagccga ggccctgggt
     3781 gccgccaaga agctgcagcc tgcacagaca gccgccaaga acctcatcat cttcctgggc
     3841 gatgggatgg gggtgtctac ggtgacagct gccaggatcc taaaagggca gaagaaggac
     3901 aaactggggc ctgagatacc cctggccatg gaccgcttcc catatgtggc tctgtccaag
     3961 acatacaatg tagacaaaca tgtgccagac agtggagcca cagccacggc ctacctgtgc
     4021 ggggtcaagg gcaacttcca gaccattggc ttgagtgcag ccgcccgctt taaccagtgc
     4081 aacacgacac gcggcaacga ggtcatctcc gtgatgaatc gggccaagaa agcagggaag
     4141 tcagtgggag tggtaaccac cacacgagtg cagcacgcct cgccagccgg cacctacgcc
     4201 cacacggtga accgcaactg gtactcggac gccgacgtgc ctgcctcggc ccgccaggag
     4261 gggtgccagg acatcgctac gcagctcatc tccaacatgg acattgacgt gatcctaggt
     4321 ggaggccgaa agtacatgtt tcgcatggga accccagacc ctgagtaccc agatgactac
     4381 agccaaggtg ggaccaggct ggacgggaag aatctggtgc aggaatggct ggcgaagcgc
     4441 cagggtgccc ggtatgtgtg gaaccgcact gagctcatgc aggcttccct ggacccgtct
     4501 gtgacccatc tcatgggtct ctttgagcct ggagacatga aatacgagat ccaccgagac
     4561 tccacactgg acccctccct gatggagatg acagaggctg ccctgcgcct gctgagcagg
     4621 aacccccgcg gcttcttcct cttcgtggag ggtggtcgca tcgaccatgg tcatcatgaa
     4681 agcagggctt accgggcact gactgagacg atcatgttcg acgacgccat tgagagggcg
     4741 ggccagctca ccagcgagga ggacacgctg agcctcgtca ctgccgacca ctcccacgtc
     4801 ttctccttcg gaggctaccc cctgcgaggg agctccatct tcgggctggc ccctggcaag
     4861 gcccgggaca ggaaggccta cacggtcctc ctatacggaa acggtccagg ctatgtgctc
     4921 aaggacggcg cccggccgga tgttaccgag agcgagagcg ggagccccga gtatcggcag
     4981 cagtcagcag tgcccctgga cgaagagacc cacgcaggcg aggacgtggc ggtgttcgcg
     5041 cgcggcccgc aggcgcacct ggttcacggc gtgcaggagc agaccttcat agcgcacgtc
     5101 atggccttcg ccgcctgcct ggagccctac accgcctgcg acctggcgcc ccccgccggc
     5161 accaccgacg ccgcgcaccc gggtggcgga ggactgaacg acatcttcga ggctcagaaa
     5221 atcgaatggc acgaacatca ccatcaccat caccatcacc attaaggccg gcctgtagtc
     5281 actggcacaa cgcgttaccc ggtaagccaa tcgggtatac acggtcgtca tactgcagac
     5341 agggttcttc tactttgcaa gatagtcttg agtagtaaaa taaatagata gagaaaatca
     5401 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     5461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatca
     5521 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5581 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     5641 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5701 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5761 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5881 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     5941 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     6001 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     6061 tacgacaagg tgaggaacta aaccatgacc gagtacaagc ccacggtgcg cctcgccacc
     6121 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     6181 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     6241 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     6301 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     6361 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     6421 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     6481 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     6541 cgcgccgggg tgcccgcctt cctggagaca tccgcgcccc gcaacctccc cttctacgag
     6601 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     6661 atgacccgca agcccggtgc ctgacacgtg ctacgagatt tcgattccac cgccgccttc
     6721 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     6781 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     6841 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     6901 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     6961 agctagagct tggcgtaatc atggtcatta ccaatgctta atcagtgagg cacctatctc
     7021 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7081 gatacgggag ggcttaccat ctggccccag cgctgcgatg ataccgcgag aaccacgctc
     7141 accggctccg gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7201 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7261 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc
     7321 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7381 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7441 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7501 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7561 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7621 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7681 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7741 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7801 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatat tcttcctt
//
