LOCUS       Exported                6967 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-MICB-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6967)
  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 6967)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6967
                     /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"
     CDS             4309..4353
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4354..4371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4511..4528)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4517..4628
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4746..4766)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4751..5080
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4931..5066
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4993..5012
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5128..5175
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5194..5793
                     /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(5194..5213)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5690..5710
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5923..6044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5960..5979)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6014..6033
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(6097..6957)
                     /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     6720..6739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6958..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 tgaacctcac
     2761 tctctgagat acaacctgat ggtgctgagc caggacgaga gcgtgcagtc tggatttctg
     2821 gccgaaggac acctggacgg ccagcctttc ctgagatacg acagacagaa gcggagagcc
     2881 aagcctcaag gacagtgggc tgaagatgtg ctgggcgcca agacctggga taccgagaca
     2941 gaggatctga ccgagaacgg ccaggacctg agaagaaccc tgacacacat caaggaccag
     3001 aaaggcggcc tgcacagcct gcaagagatc agagtgtgcg agatccacga ggacagcagc
     3061 acaagaggca gccggcactt ttactacgac ggcgagctgt tcctgagcca gaacctggaa
     3121 acccaagagt ccaccgtgcc tcagtctagc agagcccaga cactggccat gaacgtgacc
     3181 aatttctgga aagaggacgc catgaagacc aagacacact acagagccat gcaggccgac
     3241 tgcctgcaga agctgcagag atatctgaag tccggcgtgg ccatcagacg gacagtgcct
     3301 ccaatggtca acgtgacctg ttctgaggtg tccgagggca acatcaccgt gacatgcaga
     3361 gccagcagct tctaccccag aaatatcacc ctgacctggc ggcaggatgg cgtgtccctg
     3421 tctcataata cccagcagtg gggcgacgtg ctgcctgatg gcaatggcac atatcagacc
     3481 tgggtcgcca ccagaatccg gcagggcgaa gaacagcggt tcacctgtta catggaacac
     3541 agcggcaacc acggcacaca ccctgtgcca tctggaaaag tgctggtgct gcagtcccag
     3601 cggacagatg gcgcgccggg cggcgacaag acccacacct gccccccctg cccagctcca
     3661 gaactgctgg gagggccttc cgtgttcctg ttccccccca agcccaagga caccctgatg
     3721 atcagcagga cccccgaggt gacctgcgtg gtggtggccg tgtcccacga ggacccagag
     3781 gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccaga
     3841 gaggagcagt acaacagcac ctacagggtg gtgtccgtgc tgaccgtgct gcaccaggac
     3901 tggctgaacg gcaaagaata caagtgcaaa gtctccaaca aggccctggc tgccccaatc
     3961 gaaaagacaa tcagcaaggc caagggccag ccacgggagc cccaggtgta caccctgccc
     4021 cccagccggg aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc
     4081 taccccagcg atatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag
     4141 accacccccc cagtgctgga cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg
     4201 gacaagtcca ggtggcagca gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg
     4261 cacaaccact acacccagaa gtccctgagc ctgagccccg gcggcagcgg tcttaatgat
     4321 attttcgagg ctcagaagat tgagtggcat gaacaccacc accatcacca ctaaggccgg
     4381 cctgtagtca ctggcacaac gcgttacccg gtaagccaat cgggtataca cggtcgtcat
     4441 actgcagaca gggttcttct actttgcaag atagtcttga gtagtaaaat aaatagatag
     4501 agaaaatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4561 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4621 attgcatcac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4681 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     4741 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4801 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4861 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4921 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4981 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     5041 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     5101 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     5161 atagtataat acgacaaggt gaggaactaa accatgaccg agtacaagcc cacggtgcgc
     5221 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     5281 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     5341 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     5401 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     5461 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5521 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     5581 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5641 gcggccgagc gcgccggggt gcccgccttc ctggagacat ccgcgccccg caacctcccc
     5701 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5761 acctggtgca tgacccgcaa gcccggtgcc tgacacgtgc tacgagattt cgattccacc
     5821 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5881 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5941 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6001 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     6061 tcgacctcta gctagagctt ggcgtaatca tggtcattac caatgcttaa tcagtgaggc
     6121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6181 gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga
     6241 accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg
     6301 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6361 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat
     6421 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6481 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6541 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6601 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6661 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6721 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6781 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6841 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6901 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt
     6961 cttcctt
//
