LOCUS       sequence-302985-        8059 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 8059)
  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 8059)
  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-SEMA4A-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8059
                     /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             5401..5445
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5446..5463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5603..5620)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5609..5720
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5838..5858)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5843..6172
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6023..6158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6085..6104
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6220..6267
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6286..6885
                     /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(6286..6305)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6782..6802
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7015..7136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7052..7071)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7106..7125
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7189..7980,7981..8049))
                     /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     7812..7831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8050..8059,1..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 tggtggacaa
     2761 ggacctatgc ctagagtgcg gtattacgcc ggcgacgaaa gaagggccct gagcttcttt
     2821 caccagaagg gcctgcagga cttcgacaca ctgctgctga gcggagatgg caacacactg
     2881 tatgtgggcg ccagagaagc cattctggcc ctggatattc aggaccctgg cgtgcccaga
     2941 ctgaagaaca tgattccttg gcctgccagc gaccggaaga aaagcgagtg cgccttcaag
     3001 aagaagtcca acgagacaca gtgcttcaac ttcatcagag tgctggtgtc ctacaacgtg
     3061 acccacctgt acacctgtgg cacctttgcc ttttctcccg cctgcacctt catcgagctg
     3121 caggattctt acctgctgcc tatcagcgag gacaaagtga tggaaggcaa gggccagtct
     3181 cctttcgacc ctgctcacaa acataccgcc gtgctggtgg atggcatgct gtactccggc
     3241 accatgaaca actttctggg cagcgagccc atcctgatga gaacactggg aagccagcct
     3301 gtgctgaaaa ccgacaactt cctgagatgg ctgcaccacg atgccagctt tgtggccgcc
     3361 attcctagca cacaggtggt gtactttttc ttcgaggaaa ccgccagcga gttcgacttc
     3421 ttcgagagac tgcacaccag cagagtggcc agagtgtgca agaatgatgt cggcggagag
     3481 aagctgctgc agaagaagtg gaccaccttc ctgaaggccc agctgctgtg tactcagcct
     3541 ggccagctgc ctttcaacgt gatcagacat gctgtgctgc tgcccgccga ttctcctaca
     3601 gctcctcaca tctacgccgt gttcacctct cagtggcaag tcggcggaac aagatcttcc
     3661 gccgtgtgtg ccttcagcct gctggacatc gagagagtgt tcaagggcaa gtacaaagag
     3721 ctgaacaaag aaaccagccg gtggacaacc tacagaggcc ccgagacaaa tcctagacct
     3781 ggctcttgta gcgtgggccc tagctctgat aaggccctga cctttatgaa ggaccacttc
     3841 ctcatggacg agcaggtcgt gggaacacca ctgctggtta agagcggcgt cgagtacaca
     3901 agactggccg tggaaacagc ccagggcctc gatggacaca gccacctggt tatgtacctg
     3961 ggcacaacaa ccggaagcct gcacaaagcc gtggtgtctg gcgattcttc tgcccacctg
     4021 gtggaagaga tccagctgtt ccctgatcct gagcctgtgc ggaatctgca gcttgctcct
     4081 acacagggcg ccgtgtttgt gggattttct ggcggagttt ggcgggtgcc aagagccaat
     4141 tgcagcgtgt acgagagctg cgtggactgc gttctggcta gagatcctca ctgtgcttgg
     4201 gaccctgaga gcagaacctg ttgtctgctg tctgccccta acctgaactc ctggaagcag
     4261 gacatggaaa ggggcaatcc tgagtgggcc tgtgcctctg gacctatgag tagaagcctg
     4321 aggccacaga gcagacccca gatcatcaaa gaggtgctgg ctgtccccaa cagcatcctg
     4381 gaactgcctt gtcctcatct gagcgccctg gcctcttact attggagcca tggacctgct
     4441 gctgtgccag aggccagctc tacagtgtac aatggcagcc tgctcctgat cgtgcaggat
     4501 ggcgttggcg gactgtatca gtgttgggcc accgagaacg gcttcagcta ccctgtgatc
     4561 agctactggg tcgacagcca ggatcagaca ctggctctcg atcctgaact ggccggcatt
     4621 cccagagaac acgtgaaggt gcccctgaca agagttagtg gcggagctgc tctggctgcc
     4681 cagcagtctt attggcctca tggcgcgccg ggcggcgaca agacccacac ctgccccccc
     4741 tgcccagctc cagaactgct gggagggcct tccgtgttcc tgttcccccc caagcccaag
     4801 gacaccctga tgatcagcag gacccccgag gtgacctgcg tggtggtggc cgtgtcccac
     4861 gaggacccag aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag
     4921 accaagccca gagaggagca gtacaacagc acctacaggg tggtgtccgt gctgaccgtg
     4981 ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aagtctccaa caaggccctg
     5041 gctgccccaa tcgaaaagac aatcagcaag gccaagggcc agccacggga gccccaggtg
     5101 tacaccctgc cccccagccg ggaggagatg accaagaacc aggtgtccct gacctgtctg
     5161 gtgaagggct tctaccccag cgatatcgcc gtggagtggg agagcaacgg ccagcccgag
     5221 aacaactaca agaccacccc cccagtgctg gacagcgacg gcagcttctt cctgtacagc
     5281 aagctgaccg tggacaagtc caggtggcag cagggcaacg tgttcagctg cagcgtgatg
     5341 cacgaggccc tgcacaacca ctacacccag aagtccctga gcctgagccc cggcggcagc
     5401 ggtcttaatg atattttcga ggctcagaag attgagtggc atgaacacca ccaccatcac
     5461 cactaaggcc ggcctgtagt cactggcaca acgcgttacc cggtaagcca atcgggtata
     5521 cacggtcgtc atactgcaga cagggttctt ctactttgca agatagtctt gagtagtaaa
     5581 ataaatagat agagaaaatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     5641 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     5701 taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct tttgatttat
     5761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5821 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     5881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     5941 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     6001 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6061 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     6121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     6181 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     6241 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatgac cgagtacaag
     6301 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     6361 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     6421 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     6481 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     6541 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     6601 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     6661 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     6721 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac atccgcgccc
     6781 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     6841 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat
     6901 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     6961 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     7021 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     7081 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     7141 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt
     7201 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7261 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat
     7321 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg
     7381 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7441 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7501 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7561 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7621 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7681 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7741 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7801 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7861 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7921 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7981 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8041 aatactcata ttcttcctt
//
