LOCUS       sequence-410006-        8719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8719)
  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 8719)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2020.07"; journalName: "Cell"; date: "2020-08-20- 
            20"; volume: "182"; issue: "4"; pages: "1027-1043.e17"
COMMENT     Sequence Label: pD649-HAsp-ROBO3-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             6061..6105
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6106..6123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6263..6280)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6269..6380
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6498..6518)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6503..6832
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6683..6818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6745..6764
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6880..6927
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6946..7545
                     /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(6946..6965)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7442..7462
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7675..7796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7712..7731)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7766..7785
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7849..8640,8641..8709))
                     /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     8472..8491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8710..8719,1..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 cggccgccgg cgatatcagc
     2761 aattcttctg aactgctgct gggcttcaac agcagcctgg ccgctctgaa tcacaccttg
     2821 cttccacctg gcgaccccag cctgaatgga agtagagtgg gacccgagga cgccatgcct
     2881 agaatcgttg agcagcctcc tgacctgctg gtgtccagag gcgaacctgc tacactgcct
     2941 tgcagagccg aaggcagacc cagacctaac atcgagtggt acaagaacgg cgccagagtg
     3001 gccacagtgc gcgaagatcc tagagcacat aggctgctgc tgcctagcgg agccctgttc
     3061 ttcccaagaa tcgtgcatgg cagacgggcc agacctgacg aaggcgtgta cacttgcgtg
     3121 gccagaaact atctgggcgc tgccgcttcc agaaacgcct ctctggaagt ggccgtgctg
     3181 cgggacgatt tcagacagtc tcctggcaat gtggtggtgg ccgtgggaga acctgctgtg
     3241 ctggaatgtg tgcctcctag aggacaccct gagccttccg tgtcttggag aaaggatggc
     3301 gccagactga aagaggaaga gggcagaatc accatcagag gcggcaagct gatgatgagc
     3361 cacacactga agtccgacgc cgggatgtac gtgtgtgtgg cctctaatat ggccggcgag
     3421 agagaatctg ccgccgctga agtgatggtg ctggaaagac ccagcttcct gagaaggccc
     3481 gtgaaccagg ttgtgctggc tgatgcccct gtgaccttcc tgtgtgaagt gaagggcgac
     3541 cctccaccta gactgagatg gcggaaagag gatggcgagc tgcctaccgg cagatacgag
     3601 atcagatccg accactctct gtggatcggc catgtgtctg ccgaggacga gggcacatat
     3661 acctgtgtgg ccgagaactc tgtggggaga gctgaggctt ctggcagcct gtctgtgcat
     3721 gtgcctccac agctggtcac ccagcctcag gatcaaatgg ctgcccctgg cgagtctgtg
     3781 gcctttcagt gtgaaacaaa gggcaatcct cctcctgcca tcttttggca gaaagagggc
     3841 tcccaggtcc tgctgttccc tagccaatct ctgcagccca ccggccgctt ttctgtgtca
     3901 ccaagaggcc agctgaacat caccgctgtg caaagaggcg acgccggcta ctatgtgtgt
     3961 caggctgttt ctgtggccgg cagcattctg gccaaggctc tgctggaaat caagggcgct
     4021 tccctggatg gactgcctcc agtgattctt cagggcccag ccaatcagac actggtgctg
     4081 ggaagtagcg tgtggctgcc ctgtagagtg accggcaatc cacagcctag cgtgcggtgg
     4141 aagaaagacg gacaatggct gcagggcgac gacctgcagt ttaagaccat ggccaacggc
     4201 accctgtata tcgccaacgt gcaagagatg gacatggggt tctactcttg tgtggccaag
     4261 agcagcacag gcgaggctac ttggagcggc tggctgaaga tgagagagga ttggggcgtc
     4321 agccccgatc ctcctacaga gcctagtagt cctcctggcg ctccttctca gcctgtggtc
     4381 accgagatca ccaagaacag catcaccctg acctggaagc ctaatcctca gacaggcgcc
     4441 gctgtgacct cctacgtgat cgaggctttt tctcccgccg ctggcaatac ctggcggaca
     4501 gttgctgatg gcgtgcagct ggaaacccac accgtgtctg gactgcagcc taacaccatc
     4561 tacctgttcc tcgtcagagc cgttggagcc tggggactga gtgaaccctc tcctgtgtcc
     4621 gaacctgtgc ggacccaaga cagctctcct agcagacccg tggaagatcc ttggagagga
     4681 cagcagggac ttgccgaggt ggccgttaga ctgcaagagc caattgtgct gggacccaga
     4741 acactccagg tgtcctggac agtggatggc cctgtgcaac tggtgcaggg ctttagagtc
     4801 tcttggagag tggctggacc tgaaggcggc tcttggacca tgctggatct gcagtctcca
     4861 agccagcagt ccaccgtgct tagaggactg ccacctggca cacagatcca gatcaaggtg
     4921 caggcccagg gccaagaagg actgggagct gaatctctga gcgtgacccg cagcatccca
     4981 gaggaagctc catctggacc tccacagggt gttgccgttg cacttggcgg agatggcaac
     5041 agctctatca ccgtgtcctg ggaacctcca ctgcctagcc agcagaatgg cgtgatcacc
     5101 gagtatcaga tctggtgcct gggcaacgag agccggttcc acctgaatag atctgccgct
     5161 ggatgggcca gatccgccat gctgagagga cttgttcccg gcctgctgta cagaacactg
     5221 gtggccgctg ctacatctgc tggcgtggga gttccttctg ctcctgtgct ggttcagctg
     5281 ccttctcctc ctgatctgga acccggcctg gaagttggag ctggccttgc tgttagactg
     5341 gcccgggttc tgagagaacc tggcgcgccg ggcggcgaca agacccacac ctgccccccc
     5401 tgcccagctc cagaactgct gggagggcct tccgtgttcc tgttcccccc caagcccaag
     5461 gacaccctga tgatcagcag gacccccgag gtgacctgcg tggtggtggc cgtgtcccac
     5521 gaggacccag aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag
     5581 accaagccca gagaggagca gtacaacagc acctacaggg tggtgtccgt gctgaccgtg
     5641 ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aagtctccaa caaggccctg
     5701 gctgccccaa tcgaaaagac aatcagcaag gccaagggcc agccacggga gccccaggtg
     5761 tacaccctgc cccccagccg ggaggagatg accaagaacc aggtgtccct gacctgtctg
     5821 gtgaagggct tctaccccag cgatatcgcc gtggagtggg agagcaacgg ccagcccgag
     5881 aacaactaca agaccacccc cccagtgctg gacagcgacg gcagcttctt cctgtacagc
     5941 aagctgaccg tggacaagtc caggtggcag cagggcaacg tgttcagctg cagcgtgatg
     6001 cacgaggccc tgcacaacca ctacacccag aagtccctga gcctgagccc cggcggcagc
     6061 ggtcttaatg atattttcga ggctcagaag attgagtggc atgaacacca ccaccatcac
     6121 cactaaggcc ggcctgtagt cactggcaca acgcgttacc cggtaagcca atcgggtata
     6181 cacggtcgtc atactgcaga cagggttctt ctactttgca agatagtctt gagtagtaaa
     6241 ataaatagat agagaaaatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6301 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6361 taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct tttgatttat
     6421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6481 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     6541 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     6601 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     6661 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6721 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     6781 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     6841 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     6901 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatgac cgagtacaag
     6961 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     7021 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     7081 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     7141 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     7201 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     7261 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     7321 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     7381 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac atccgcgccc
     7441 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     7501 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat
     7561 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     7621 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     7681 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     7741 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     7801 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt
     7861 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7921 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat
     7981 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg
     8041 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     8101 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     8161 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     8221 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8281 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8341 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8401 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8461 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8521 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8581 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8641 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8701 aatactcata ttcttcctt
//
