LOCUS       Exported                8620 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-ROBO2-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8620)
  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 8620)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8620
                     /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             5962..6006
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6007..6024
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6164..6181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6170..6281
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6399..6419)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6404..6733
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6584..6719
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6646..6665
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6781..6828
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6847..7446
                     /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(6847..6866)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7343..7363
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7576..7697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7613..7632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7667..7686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(7750..8610)
                     /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     8373..8392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8611..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 cggccgccag tagactgaga
     2761 caagaggact tccctcctcg gatcgtggaa caccctagcg acgtgatcgt gtctaagggc
     2821 gagcccacca cactgaactg taaagccgaa ggcagaccca cacctaccat cgagtggtac
     2881 aaggacggcg agagagtgga aaccgacaag gacgaccctc ggagccatag aatgctgctg
     2941 cctagcggca gcctgttctt cctgagaatc gtgcacggca gaagaagcaa gcctgacgag
     3001 ggctcttacg tgtgcgtggc cagaaattat ctgggcgaag ccgtgtccag aaacgcctct
     3061 ctggaagtgg ccctgctgag ggacgacttc agacagaatc ctaccgatgt ggtggtggcc
     3121 gctggcgaac ctgccattct tgagtgtcag cctccaagag gacaccccga gcctaccatc
     3181 tactggaaga aagacaaagt gcggatcgac gacaaagagg aacggatcag catcagaggc
     3241 ggcaagctga tgatcagcaa caccagaaag tccgacgccg gcatgtacac ctgtgtgggc
     3301 acaaacatgg tcggagagag agactctgat cccgccgagc tgaccgtgtt cgaaagaccc
     3361 acatttctgc ggaggcccat caaccaggtg gtgctggaag aagaggccgt cgagtttaga
     3421 tgccaggtgc agggcgatcc tcagcctaca gtgcggtgga aaaaggacga cgccgatctg
     3481 cccagaggca gatacgacat caaggacgat tacaccctgc ggatcaagaa aaccatgagc
     3541 accgacgagg gcacctacat gtgtatcgcc gagaacagag tgggcaagat ggaagcctct
     3601 gccacactga cagttcgggc ccctcctcag tttgtcgtca gacccagaga tcagatcgtg
     3661 gcccagggca gaaccgtgac ctttccatgc gagacaaagg gcaatcccca gcctgccgtg
     3721 ttttggcaga aagagggcag ccagaacctg ctgttcccca atcagcctca gcagcccaat
     3781 agcagatgta gcgtgtcacc taccggcgac ctgaccatca ccaacatcca gagaagcgac
     3841 gctggctact acatctgtca ggctctgacc gtggccggct ctattctggc taaagcccag
     3901 ctggaagtga ccgacgtgct gaccgataga cctcctccaa tcattctgca gggcccagcc
     3961 aatcagacac tggccgttga tggaaccgct ctgctgaagt gcaaagccac aggcgatcca
     4021 ctgcctgtga tcagctggct gaaagaaggc ttcacattcc ccggcagaga tcccagagcc
     4081 acaatccaag aacagggcac actgcagatt aagaacctgc gcatcagcga caccggcacc
     4141 tatacttgtg tggccacctc tagcagcggc gagacatctt ggagtgccgt gctggatgtg
     4201 acagagagcg gcgccaccat cagcaagaac tacgatctga gcgacctgcc tggacctcct
     4261 agcaagcctc aagtgactga tgtgaccaag aatagcgtga ccctgagctg gcagcctggc
     4321 acacctggaa ctctgcctgc ctctgcctac atcattgagg ccttcagcca gtccgtgtcc
     4381 aactcttggc agacagtggc caaccacgtg aaaaccacac tgtacaccgt cagaggcctg
     4441 cggcctaaca ccatctacct gttcatggtc cgagccatca atcctcaggg cctgagcgat
     4501 ccatctccta tgtctgatcc tgtgcggacc caggacatct ctccaccagc tcaaggcgtg
     4561 gaccacagac aggttcagaa agaactggga gatgtgctcg tgcggctgca caatcctgtg
     4621 gtgctgaccc ctaccaccgt gcaagtgacc tggacagtgg acagacagcc ccagttcatc
     4681 cagggctaca gagtgatgta cagacagacc agcggactgc aggccaccag tagctggcag
     4741 aatctggatg ccaaggtgcc aaccgaaaga tccgccgtgc tcgtgaacct gaagaaaggc
     4801 gtgacctacg agatcaaagt ccggccttac ttcaacgagt tccaaggcat ggacagcgag
     4861 agcaagaccg tgcggacaac agaggaagct cctagcgctc ctccacagag cgtgacagtg
     4921 ctgacagtgg gcagctacaa cagcaccagc atctccgttt cttgggaccc tcctccacct
     4981 gaccaccaga acggcatcat ccaagagtac aagatctggt gcctgggcaa cgagacacgg
     5041 ttccacatca acaagactgt ggacgccgcc atcagaagcg tgatcatcgg aggactgttc
     5101 ccaggcatcc agtacagagt tgaggtggcc gcatcaacat ctgccggcgt gggagtgaaa
     5161 agcgagcccc agcctatcat catcggcaga cggaatgagg tggtcatcac cgagaacaac
     5221 aacagcatca cagagcagat caccgacgtg gtcaagcagc ctggcgcgcc gggcggcgac
     5281 aagacccaca cctgcccccc ctgcccagct ccagaactgc tgggagggcc ttccgtgttc
     5341 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc
     5401 gtggtggtgg ccgtgtccca cgaggaccca gaggtgaagt tcaactggta cgtggacggc
     5461 gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg
     5521 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc
     5581 aaagtctcca acaaggccct ggctgcccca atcgaaaaga caatcagcaa ggccaagggc
     5641 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac
     5701 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg
     5761 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac
     5821 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac
     5881 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
     5941 agcctgagcc ccggcggcag cggtcttaat gatattttcg aggctcagaa gattgagtgg
     6001 catgaacacc accaccatca ccactaaggc cggcctgtag tcactggcac aacgcgttac
     6061 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc
     6121 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct
     6181 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6241 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct
     6301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6361 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     6421 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6481 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6541 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6601 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6661 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     6721 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     6781 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     6841 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     6901 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     6961 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     7021 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     7081 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     7141 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     7201 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     7261 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     7321 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     7381 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     7441 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7501 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7561 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     7621 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7681 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     7741 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7801 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7861 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     7921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     8041 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     8101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8161 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     8221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8341 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8581 aagggcgaca cggaaatgtt gaatactcat attcttcctt
//
