LOCUS       Exported                9106 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-SEMA5B-Fc(DAPA)-AviTag-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9106)
  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 9106)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9106
                     /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             6448..6492
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6493..6510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6650..6667)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6656..6767
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6885..6905)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6890..7219
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7070..7205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7132..7151
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7267..7314
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7333..7932
                     /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(7333..7352)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7829..7849
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    8062..8183
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8099..8118)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8153..8172
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(8236..9096)
                     /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     8859..8878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9097..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 cggccgcctt gtctcttgtt
     2761 agaggcctgc ttccttgcct gccaccaggc gctagaacag ccgagggacc tattatggtg
     2821 ctggctggac ctctggccgt gtctctgctt ctgccttctc tgaccctgct ggtgtctcac
     2881 ctgtctagca gccaggatgt gtccagcgag cctagctctg agcagcagct gtgtgccctg
     2941 agcaagcacc ctacagtggc cttcgaagat ctgcagccct gggtgtccaa cttcacatac
     3001 cctggggcca gagacttctc ccagctggct cttgatccca gcggcaacca gctgattgtg
     3061 ggcgccagaa actacctgtt cagactgagc ctggccaacg tgtcactgct gcaggctaca
     3121 gagtgggcca gcagcgagga taccagaaga agctgtcaga gcaagggcaa gaccgaggaa
     3181 gagtgccaga actacgtgcg ggtgctgatc gtggccggca gaaaggtttt catgtgcggc
     3241 accaacgctt tcagccccat gtgcaccagc agacaagtgg gcaacctgag ccggaccatc
     3301 gagaagatca acggcgtggc cagatgtccc tacgatccca gacacaatag caccgccgtg
     3361 atcagctctc agggcgaact gtatgccgcc accgtgatcg atttcagcgg cagagatccc
     3421 gccatctaca gaagcctcgg atctggccct ccactgagaa cagcccagta caacagcaag
     3481 tggctgaacg agcccaactt cgtggccgcc tatgacatcg gcctgttcgc ctacttcttc
     3541 ctgcgggaaa acgccgtgga acacgactgt ggcagaaccg tgtatagcag agtggccaga
     3601 gtgtgcaaga acgacgtcgg cggcagattc ctgctggaag atacctggac cacctttatg
     3661 aaggcccggc tgaactgcag cagacctggc gaggtgccct tctactacaa cgagctgcag
     3721 agcgccttcc atctgcctga gcaggatctg atctacggcg tgttcaccac caacgtgaac
     3781 tctatcgccg cctctgccgt gtgcgccttt aatctgtctg ccatcagcca ggcctttaac
     3841 ggccccttca gataccaaga gaaccccaga gctgcctggc tgcctatcgc caatcctatt
     3901 cctaacttcc agtgcggcac actgcccgag acaggcccca atgagaatct gaccgagaga
     3961 agcctgcagg acgcccagag actgttcctg atgtctgaag ccgtgcagcc cgtgacacct
     4021 gagccttgtg tgacacagga tagcgtgcgg ttctcccacc tggtggtgga tctggtgcag
     4081 gccaaagaca ccctgtacca cgtgctgtac atcggcacag agagcggcac aatcctgaag
     4141 gccctgtcta ccgccagcag atccctgcac ggatgctacc tggaagaact gcacgtgctg
     4201 ccacctggca gaagagagcc tctgagatcc ctgagaatcc tgcacagcgc cagggctctg
     4261 tttgtgggac tgagagatgg cgtgctgaga gtgcccctgg aaagatgtgc cgcctacaga
     4321 tcacagggcg cctgtctggg agctagagat ccttactgtg gctgggacgg caagcagcag
     4381 agatgtagca ccctggaaga ttccagcaac atgagcctgt ggacccagaa catcaccgcc
     4441 tgtcctgtgc ggaacgtgac acgagatggc ggctttggac cttggagtcc ttggcagcct
     4501 tgcgagcatc tggacggcga taatagcggc tcctgcctgt gtagagccag aagctgcgat
     4561 agccccagac ctagatgtgg cggcctggat tgtctgggcc ctgccatcca tatcgccaac
     4621 tgctccagaa acggcgcctg gacaccttgg tcatcttggg ccctgtgtag cacctcttgc
     4681 ggcatcggat ttcaagtccg gcagcggagc tgcagcaacc ctgctcctag acatggcggc
     4741 agaatctgcg tgggcaagtc ccgggaagag agattctgca acgagaacac cccttgtcca
     4801 gtgcctatct tctgggcctc ttggggcagc tggtccaagt gcagctctaa ttgtggcggc
     4861 ggaatgcaga gcagaagaag ggcctgcgag aacggcaata gctgtctcgg atgcggcgtg
     4921 gaattcaaga cctgcaatcc tgagggctgc cccgaagtgc ggagaaacac accttggact
     4981 ccatggctgc ccgtgaacgt tacacaaggc ggagccagac aagagcagcg gttccggttt
     5041 acctgtagag cccctctggc tgatcctcac ggcctgcagt tcggaagaag aagaaccgag
     5101 acacggacct gtcctgccga tggcagcggc tcttgtgata cagatgccct ggtggaagtg
     5161 ctgctgagaa gcggctctac aagccctcac acagtgtctg gtggatgggc tgcttgggga
     5221 ccttggtcca gctgtagcag agattgcgag ctgggcttca gagtgcggaa gcggacctgt
     5281 accaatccag agcctaggaa cggcggcctg ccatgtgttg gagatgccgc cgaataccag
     5341 gactgcaacc ctcaggcctg tccagttaga ggcgcctggt cttgttggac ctcttggagc
     5401 ccttgttccg cctcttgtgg cggaggccac taccagagaa caagaagctg cacaagcccc
     5461 gcaccttctc caggcgagga tatttgcctg ggcctgcata ctgaagaggc cctgtgcgca
     5521 acacaggctt gtccagaagg atggtcccct tggagcgagt ggtctaagtg cacagatgac
     5581 ggcgcccaga gcagatctag gcactgcgag gaactgctgc ctggatcttc tgcctgtgcc
     5641 ggcaattcta gccagagccg gccttgtcca tacagcgaga tcccagtgat cctgcctgcc
     5701 agctctatgg aagaggccac agattgcgcc ggcttcaacc tgatccatgg cgcgccgggc
     5761 ggcgacaaga cccacacctg ccccccctgc ccagctccag aactgctggg agggccttcc
     5821 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg
     5881 acctgcgtgg tggtggccgt gtcccacgag gacccagagg tgaagttcaa ctggtacgtg
     5941 gacggcgtgg aggtgcacaa cgccaagacc aagcccagag aggagcagta caacagcacc
     6001 tacagggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaagaatac
     6061 aagtgcaaag tctccaacaa ggccctggct gccccaatcg aaaagacaat cagcaaggcc
     6121 aagggccagc cacgggagcc ccaggtgtac accctgcccc ccagccggga ggagatgacc
     6181 aagaaccagg tgtccctgac ctgtctggtg aagggcttct accccagcga tatcgccgtg
     6241 gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc agtgctggac
     6301 agcgacggca gcttcttcct gtacagcaag ctgaccgtgg acaagtccag gtggcagcag
     6361 ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag
     6421 tccctgagcc tgagccccgg cggcagcggt cttaatgata ttttcgaggc tcagaagatt
     6481 gagtggcatg aacaccacca ccatcaccac taaggccggc ctgtagtcac tggcacaacg
     6541 cgttacccgg taagccaatc gggtatacac ggtcgtcata ctgcagacag ggttcttcta
     6601 ctttgcaaga tagtcttgag tagtaaaata aatagataga gaaaatcagc ctcgactgtg
     6661 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     6721 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcaca acactcaacc
     6781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6841 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     6901 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     6961 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     7021 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     7081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     7141 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     7201 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     7261 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     7321 aggaactaaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     7381 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     7441 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     7501 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     7561 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     7621 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     7681 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     7741 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     7801 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     7861 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     7921 cccggtgcct gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     7981 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     8041 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     8101 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     8161 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     8221 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     8281 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     8341 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga
     8401 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     8461 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     8521 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt
     8581 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     8641 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     8701 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     8761 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     8821 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     8881 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     8941 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     9001 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     9061 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctt
//
