LOCUS       Exported                7708 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    pD649-HAsp-SIGLEC10-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7708)
  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 7708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7708
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5050..5094
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5095..5112
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5252..5269)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5258..5369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5487..5507)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5492..5821
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5672..5807
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5734..5753
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5869..5916
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5935..6534
                     /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(5935..5954)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6431..6451
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6664..6785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6701..6720)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6755..6774
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(6838..7698)
                     /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     7461..7480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7699..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 ggatggcaga
     2761 ttctggatca gagtgcaaga gtccgtgatg gtgcctgagg gcctgtgtat cagcgtgccc
     2821 tgcagcttca gctaccccag acaggattgg acaggcagca caccagccta cggctactgg
     2881 tttaaggccg tgaccgaaac caccaagggc gctccagtgg ccacaaacca ccagagcaga
     2941 gaagtggaaa tgagcacccg gggcagattc cagctgacag gcgatcctgc caagggcaat
     3001 tgcagcctgg tcatcagaga tgcccagatg caggacgaga gccagtactt cttcagagtg
     3061 gaacggggca gctacgtgcg gtacaacttt atgaacgacg gattcttcct gaaagtgacg
     3121 gccctgacac agaaacccga cgtgtacatc cccgagacac tggaacctgg ccagcctgtg
     3181 acagtgatct gcgtgttcaa ctgggccttc gaggaatgcc ctccaccttc cttttcttgg
     3241 actggcgccg ctctgtctag ccagggcaca aagcctacca ccagccactt tagcgtgctg
     3301 agcttcaccc ctagacctca ggaccacaac accgacctga cctgccacgt ggacttcagc
     3361 agaaaaggcg tgtcagccca gagaaccgtg cggctgagag tggcctatgc tcccagagat
     3421 ctcgtgatca gcatcagccg ggacaacacc cctgctctgg aacctcagcc tcagggcaat
     3481 gtgccttacc tggaagccca gaagggccag ttcctgagac tgctgtgtgc cgccgattct
     3541 cagcctcctg ctacactgtc ttgggtgctg cagaacagag tgctgagcag ctctcaccct
     3601 tggggcccta gaccactggg acttgaactg cctggtgtca aggccggcga cagcggcaga
     3661 tacacatgca gagccgagaa cagactgggc agccagcaga gagcactgga tctgagcgtg
     3721 cagtaccctc ctgagaacct gagagtgatg gtgtcccagg ccaaccggac cgtgctggaa
     3781 aatctcggca atggcacaag cctgcctgtg ctggaaggcc agtctctgtg tctcgtgtgt
     3841 gtgacccaca gcagccctcc agccagactc tcttggacac agcgaggaca ggtgctgtct
     3901 cctagccagc cttctgatcc tggcgtcctg gaactgccta gagtgcaggt tgagcacgag
     3961 ggcgagttca cctgtcacgc cagacatcct ctgggctctc agcacgtttc cctgagcctg
     4021 tctgtgcact acagccctaa gctgctgggc cctagctgtt cttgggaagc cgaaggcctg
     4081 cactgcagct gtagctctca agcctctcct gctcctagcc tgagatggtg gctgggcgaa
     4141 gaactgctcg agggcaatag ctcccaggac agcttcgaag tgaccccttc ttctgccgga
     4201 ccttgggcca atagctctct gtctctgcac ggcggactga gcagtggact gagactgaga
     4261 tgcgaggcct ggaatgtgca cggtgctcag tctggctcta tcctgcagct gcccgataag
     4321 aagggcctga tcagcaccgc cttcagcaat ggcgcgccgg gcggcgacaa gacccacacc
     4381 tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct gttccccccc
     4441 aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggcc
     4501 gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac
     4561 aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg
     4621 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa agtctccaac
     4681 aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca gccacgggag
     4741 ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg
     4801 acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga gagcaacggc
     4861 cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc
     4921 ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt gttcagctgc
     4981 agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcccc
     5041 ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca tgaacaccac
     5101 caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa
     5161 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg
     5221 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca
     5281 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5341 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt
     5401 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5461 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     5521 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     5581 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5641 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     5701 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     5761 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     5821 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     5881 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc
     5941 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     6001 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     6061 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     6121 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     6181 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6241 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6301 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     6361 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca
     6421 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     6481 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg
     6541 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     6601 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     6661 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6721 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6781 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta
     6841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6901 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6961 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
     7021 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7081 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7141 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7201 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7261 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7321 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7381 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7441 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7501 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7561 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7621 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7681 gaaatgttga atactcatat tcttcctt
//
