LOCUS       sequence-396946-        7147 bp    DNA     circular SYN 14-APR-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 Apr 14, 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-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      977..986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2723..2767
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2768..2785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2925..2942)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2931..3042
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3160..3180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3165..3494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3345..3480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3407..3426
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3542..3589
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3608..4207
                     /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(3608..3627)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4104..4124
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4337..4458
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4374..4393)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4428..4447
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4511..5302,5303..5371))
                     /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     5134..5153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5372..5463)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5505..5524
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5530..5549
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5635..6223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6124..6143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6377..6394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6492..6509
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6493..6509
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6965..6981
                     /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)."
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgaaaacgcg cctgctagcg ccaccatgaa gaccatcatt gccctgagct acatcttctg
      961 cctggtgttc gctgcggccg ccatggaagg cgatagacaa tacggcgacg gctacctgct
     1021 gcaggtccaa gagctggtca cagtgcaaga gggcctgtgt gtgcacgtgc cctgcagctt
     1081 tagctaccct caggatggct ggaccgacag cgatcctgtg cacggctatt ggttcagagc
     1141 cggcgacaga ccctatcagg atgcccctgt ggccaccaac aatcccgata gagaggtgca
     1201 ggccgagaca cagggcagat ttcagctgct gggcgacatc tggtccaacg actgtagcct
     1261 gagcatccgg gacgcccgga agagagataa gggcagctac ttcttccggc tggaacgggg
     1321 cagcatgaag tggtcctaca agagccagct gaactacaag accaagcagc tgagcgtgtt
     1381 cgtgacagcc ctgacacaca gacccgacat cctgatcctg ggcacactgg aatctggcca
     1441 cagcagaaac ctgacctgta gcgtgccctg ggcctgcaaa cagggaaccc ctcctatgat
     1501 ctcttggatc ggcgccagcg tgtcctctcc tggacctaca acagctagaa gcagcgtgct
     1561 gaccctgaca cctaagcctc aggatcacgg caccagcctg acctgccaag ttacacttcc
     1621 tggcaccggc gtgaccacca caagcacagt tagactggac gtgtcctatc ctccttggaa
     1681 cctgaccatg accgtgttcc agggcgacgc cacagcctct acagccctcg gaaatggcag
     1741 cagcctgtct gtgctggaag gccagtctct gagactcgtg tgcgccgtga acagcaaccc
     1801 tcctgctaga ctgagctgga ccagaggcag cctgacactg tgtcctagca gaagcagcaa
     1861 tcccggcctg ctggaactgc ctagagtgca cgttcgagat gagggcgagt tcacctgtag
     1921 agcccagaat gcccagggct cccagcacat ctctctgagc ctgagcctgc agaatgaagg
     1981 caccggaacc agcagacccg tgtctcaagt gacactggct gctggcgcgc cgggcggcga
     2041 caagacccac acctgccccc cctgcccagc tccagaactg ctgggagggc cttccgtgtt
     2101 cctgttcccc cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg
     2161 cgtggtggtg gccgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg
     2221 cgtggaggtg cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag
     2281 ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg aacggcaaag aatacaagtg
     2341 caaagtctcc aacaaggccc tggctgcccc aatcgaaaag acaatcagca aggccaaggg
     2401 ccagccacgg gagccccagg tgtacaccct gccccccagc cgggaggaga tgaccaagaa
     2461 ccaggtgtcc ctgacctgtc tggtgaaggg cttctacccc agcgatatcg ccgtggagtg
     2521 ggagagcaac ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga
     2581 cggcagcttc ttcctgtaca gcaagctgac cgtggacaag tccaggtggc agcagggcaa
     2641 cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct
     2701 gagcctgagc cccggcggca gcggtcttaa tgatattttc gaggctcaga agattgagtg
     2761 gcatgaacac caccaccatc accactaagg ccggcctgta gtcactggca caacgcgtta
     2821 cccggtaagc caatcgggta tacacggtcg tcatactgca gacagggttc ttctactttg
     2881 caagatagtc ttgagtagta aaataaatag atagagaaaa tcagcctcga ctgtgccttc
     2941 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3001 cactcccact gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc
     3061 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3121 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3241 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3301 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3361 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3421 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3481 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     3541 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     3601 ctaaaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3661 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3721 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3781 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3841 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3901 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3961 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     4021 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     4081 cttcctggag acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     4141 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     4201 tgcctgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4261 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4321 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4381 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4441 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4501 atcatggtca ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4561 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4621 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     4681 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4741 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4801 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4861 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4921 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4981 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5041 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5101 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5161 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5221 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5281 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5341 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca
     5401 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5461 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta
     5521 tacctgaata tggctcataa caccccttgc tcatgaccaa aatcccttaa cgtgagttac
     5581 gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5641 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5701 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5761 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttagcccacc acttcaagaa
     5821 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5881 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5941 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6001 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6061 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6121 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6181 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6241 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6301 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6361 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaggcgaga gtagggaact
     6421 gccaggcatc aaactaagca gaaggcccct gacggatggc ctttttgcgt ttctacaaac
     6481 tctttctgtg ttgtaaaacg acggccagtc ttaagctcgg gccccctggg cggttctgat
     6541 aacgagtaat cgttaatccg caaataacgt aaaaacccgc ttcggcgggt ttttttatgg
     6601 ggggagttta gggaaagagc atttgtcaga atatttaagg gcgcctgtca ctttgcttga
     6661 tatatgagaa ttatttaacc ttataaatga gaaaaaagca acgcacttta aataagatac
     6721 gttgcttttt cgattgatga acacctataa ttaaactatt catctattat ttatgatttt
     6781 ttgtatatac aatatttcta gtttgttaaa gagaattaag aaaataaatc tcgaaaataa
     6841 taaagggaaa atcagttttt gatatcaaaa ttatacatgt caacgataat acaaaatata
     6901 atacaaacta taagatgtta tcagtattta ttatcattta gaataaattt tgtgtcgccc
     6961 ttaattgtga gcggataaca attacgagct tcatgcacag tggcgttgac tcaatattgg
     7021 ccattagcca tattattcat tggttatata gcataaatca atattggcta ttggccattg
     7081 catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg
     7141 ccatgtt
//
