LOCUS       Exported                8722 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-ROBO1-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8722)
  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 8722)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8722
                     /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             6064..6108
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6109..6126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6266..6283)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6272..6383
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6501..6521)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6506..6835
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6686..6821
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6748..6767
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6883..6930
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6949..7548
                     /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(6949..6968)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7445..7465
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7678..7799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7715..7734)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7769..7788
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(7852..8712)
                     /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     8475..8494
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8713..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 acttattccc
     2761 gatcctgagg atgtggaacg gggcaacgat cacggcaccc ctattcctac cagcgacaac
     2821 gacgacaaca gcctgggcta cacaggcagc agactgcggc aagaggactt cccacctcgg
     2881 atcgtggaac accccagcga tctgatcgtg tccaaaggcg aacccgccac actgaactgc
     2941 aaggccgaag gcagacccac acctaccatc gagtggtaca aaggcggcga gagagtggaa
     3001 accgacaagg acgatcctcg gagccacaga atgctgctgc ctagcggcag cctgttcttc
     3061 ctgagaatcg tgcacggcag aaagagcaga cctgacgagg gcgtgtacgt gtgcgtggcc
     3121 agaaattatc tgggcgaagc cgtgtctcac aacgccagcc tggaagtggc catcctgagg
     3181 gacgacttca gacagaaccc ctccgatgtg atggtggccg tgggagaacc tgccgtgatg
     3241 gaatgccaac ctcctagagg acaccccgag cctaccatca gctggaagaa ggatggcagc
     3301 cctctggacg acaaggatga gcggatcaca atcagaggcg gcaagctgat gatcacctac
     3361 accagaaagt ccgacgccgg caaatacgtg tgtgtgggca ccaacatggt cggagagcgg
     3421 gaatctgaag tggccgagct gaccgtgctg gaaagaccca gcttcgtgaa gaggcccagc
     3481 aatctggccg tgaccgtgga tgatagcgcc gagtttaagt gcgaggccag aggcgatcct
     3541 gtgcctacag ttagatggcg gaaggacgat ggcgagctgc ccaagagcag atacgagatc
     3601 agggacgatc acaccctgaa gatccggaaa gtgacagccg gcgacatggg cagctatacc
     3661 tgtgtggccg agaatatggt cggaaaggcc gaggccagcg ccactctgac agttcaagag
     3721 cctcctcact tcgtggtcaa gcccagagat caggtggtgg ctctgggcag aaccgtgacc
     3781 tttcagtgtg aagccaccgg caatccccag cctgccatct tttggagaag agagggcagc
     3841 cagaacctgc tgttcagcta ccagcctcca cagagcagca gcagattcag cgttagccag
     3901 accggcgacc tgaccatcac caacgtgcag agatccgacg tgggctacta catctgtcag
     3961 accctgaatg tggccggcag catcatcacc aaggcctatc tggaagtgac cgacgtgatc
     4021 gccgacagac ctcctccagt gatcagacag ggccccgtga atcagaccgt ggctgtggat
     4081 ggcaccttcg tgctgtcttg tgtggccacc ggatctcccg tgcctacaat cctgtggcgg
     4141 aaagatggcg tgctggtgtc tacccaggac agcagaatca agcagctgga aaacggcgtg
     4201 ctgcagatca gatacgccaa gctgggcgac accggcagat acacctgtat cgcctctaca
     4261 cctagcggcg aggctacttg gagcgcctac attgaggtgc aagagttcgg cgtgccagtg
     4321 cagcctccta gacctacaga ccccaatctg atccccagcg ctcccagcaa gcctgaagtg
     4381 actgatgtgt cccggaacac cgtgacactg agctggcagc ccaacctgaa ttctggcgcc
     4441 acaccaacca gctacatcat cgaggccttt agccacgcca gcggcagctc ttggcagaca
     4501 gtggccgaaa acgtgaaaac cgagacaagc gccatcaagg gcctgaagcc taacgctatc
     4561 tacctgttcc tcgtgcgggc tgccaatgcc tacggcatct ctgatcctag ccagatcagc
     4621 gaccctgtga aaacccagga cgtgctgcct acatctcagg gcgtcgacca caaacaggtg
     4681 cagagagaac tgggcaacgc tgtgctgcat ctgcacaacc ctacagtgct gagcagctcc
     4741 agcatcgagg tgcactggac agtggatcag cagagccagt acatccaggg ctacaagatc
     4801 ctgtacagac ccagcggagc caaccacggc gaatctgatt ggctggtgtt cgaagtgcgg
     4861 acccctgcca agaacagcgt ggtcatcccc gatctgagaa agggcgtgaa ctatgagatc
     4921 aaggccaggc cattcttcaa cgagttccag ggcgccgaca gcgagatcaa gttcgccaag
     4981 acactggaag aggctccctc cgctcctcca caaggcgtga cagtgtccaa gaacgacggc
     5041 aatggcaccg ccatcctggt gtcatggcaa cctccacctg aggataccca gaacggcatg
     5101 gtccaagagt acaaagtgtg gtgcctgggg aacgagacac gctaccacat caacaagacc
     5161 gtggacggct ctaccttcag cgtcgtgatc ccttttctgg tgcccggcat ccggtactct
     5221 gtggaagttg ctgcttctac cggcgctggc tctggcgtga aaagcgagcc tcagttcatt
     5281 cagctggacg cccacggcaa ccctgtgtct ccagaggacc aagtgtctct ggcccagcag
     5341 atttccgacg tcgtgaagca acctggcgcg ccgggcggcg acaagaccca cacctgcccc
     5401 ccctgcccag ctccagaact gctgggaggg ccttccgtgt tcctgttccc ccccaagccc
     5461 aaggacaccc tgatgatcag caggaccccc gaggtgacct gcgtggtggt ggccgtgtcc
     5521 cacgaggacc cagaggtgaa gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc
     5581 aagaccaagc ccagagagga gcagtacaac agcacctaca gggtggtgtc cgtgctgacc
     5641 gtgctgcacc aggactggct gaacggcaaa gaatacaagt gcaaagtctc caacaaggcc
     5701 ctggctgccc caatcgaaaa gacaatcagc aaggccaagg gccagccacg ggagccccag
     5761 gtgtacaccc tgccccccag ccgggaggag atgaccaaga accaggtgtc cctgacctgt
     5821 ctggtgaagg gcttctaccc cagcgatatc gccgtggagt gggagagcaa cggccagccc
     5881 gagaacaact acaagaccac ccccccagtg ctggacagcg acggcagctt cttcctgtac
     5941 agcaagctga ccgtggacaa gtccaggtgg cagcagggca acgtgttcag ctgcagcgtg
     6001 atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgagcctgag ccccggcggc
     6061 agcggtctta atgatatttt cgaggctcag aagattgagt ggcatgaaca ccaccaccat
     6121 caccactaag gccggcctgt agtcactggc acaacgcgtt acccggtaag ccaatcgggt
     6181 atacacggtc gtcatactgc agacagggtt cttctacttt gcaagatagt cttgagtagt
     6241 aaaataaata gatagagaaa atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6301 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6361 taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat tcttttgatt
     6421 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6481 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     6541 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     6601 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6661 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6721 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     6781 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6841 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
     6901 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gaccgagtac
     6961 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     7021 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     7081 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     7141 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7201 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7261 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7321 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7381 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     7441 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7501 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga
     7561 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     7621 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     7681 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     7741 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7801 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc attaccaatg
     7861 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7921 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     7981 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     8041 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     8101 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     8161 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8221 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8281 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8341 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8401 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8461 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8521 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8581 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8641 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8701 ttgaatactc atattcttcc tt
//
