LOCUS       Exported                6757 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-CNTFR-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6757)
  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 6757)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6757
                     /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             4099..4143
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4144..4161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4301..4318)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4307..4418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4536..4556)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4541..4870
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4721..4856
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4783..4802
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4918..4965
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4984..5583
                     /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(4984..5003)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5480..5500
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5713..5834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5750..5769)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5804..5823
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(5887..6747)
                     /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     6510..6529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6748..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 cggccgccca aagacattct
     2761 cctcaagagg cccctcacgt gcagtacgag agactgggat ctgatgtgac cctgccttgt
     2821 ggcacagcca attgggatgc cgctgtgact tggagagtga acggaaccga tctggcccct
     2881 gatctgctga atggatctca gctggtgctg cacggactgg aactgggcca ttctggcctg
     2941 tacgcctgct tccacagaga ctcttggcac ctgagacacc aggtgctgct gcatgttggc
     3001 ctgcctccaa gagaacctgt gctgagctgc agaagcaaca cataccccaa gggcttctac
     3061 tgcagctggc atctgcccac acctacctac attcccaaca ccttcaacgt gaccgtgctg
     3121 catggcagca agatcatggt ctgcgagaag gaccccgctc tgaagaacag atgccacatc
     3181 cggtacatgc acctgttcag caccatcaag tacaaggtgt ccatctccgt gtctaacgcc
     3241 ctgggacaca atgccaccgc catcaccttc gacgagttca ccatcgtgaa gcccgatcct
     3301 ccagagaacg tggtggctag acctgtgcct agcaacccta gaaggctgga agtgacctgg
     3361 cagaccccta gcacatggcc tgatcctgag agcttccctg gcgcgccggg cggcgacaag
     3421 acccacacct gccccccctg cccagctcca gaactgctgg gagggccttc cgtgttcctg
     3481 ttccccccca agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg
     3541 gtggtggccg tgtcccacga ggacccagag gtgaagttca actggtacgt ggacggcgtg
     3601 gaggtgcaca acgccaagac caagcccaga gaggagcagt acaacagcac ctacagggtg
     3661 gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagaata caagtgcaaa
     3721 gtctccaaca aggccctggc tgccccaatc gaaaagacaa tcagcaaggc caagggccag
     3781 ccacgggagc cccaggtgta caccctgccc cccagccggg aggagatgac caagaaccag
     3841 gtgtccctga cctgtctggt gaagggcttc taccccagcg atatcgccgt ggagtgggag
     3901 agcaacggcc agcccgagaa caactacaag accacccccc cagtgctgga cagcgacggc
     3961 agcttcttcc tgtacagcaa gctgaccgtg gacaagtcca ggtggcagca gggcaacgtg
     4021 ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc
     4081 ctgagccccg gcggcagcgg tcttaatgat attttcgagg ctcagaagat tgagtggcat
     4141 gaacaccacc accatcacca ctaaggccgg cctgtagtca ctggcacaac gcgttacccg
     4201 gtaagccaat cgggtataca cggtcgtcat actgcagaca gggttcttct actttgcaag
     4261 atagtcttga gtagtaaaat aaatagatag agaaaatcag cctcgactgt gccttctagt
     4321 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4381 cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac cctatctcgg
     4441 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4501 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     4561 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4621 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4681 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4801 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4861 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
     4921 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     4981 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     5041 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     5101 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     5161 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     5221 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     5281 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     5341 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     5401 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     5461 ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     5521 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     5581 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5641 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5701 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5761 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5821 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5881 tggtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5941 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6001 tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg atttatcagc
     6061 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6121 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6181 gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6241 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6301 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6361 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6421 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6481 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6541 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6601 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6661 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6721 ggcgacacgg aaatgttgaa tactcatatt cttcctt
//
