LOCUS       sequence-302977-        8032 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 8032)
  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 8032)
  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-SEMA6D-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8032
                     /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             5374..5418
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5419..5436
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5576..5593)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5582..5693
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5811..5831)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5816..6145
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5996..6131
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6058..6077
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6193..6240
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6259..6858
                     /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(6259..6278)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6755..6775
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6988..7109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7025..7044)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7079..7098
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7162..7953,7954..8022))
                     /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     7785..7804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8023..8032,1..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 cggccgccgt tagctttcct
     2761 gaggatgacg agcccctgaa caccgtggac taccactaca gcagacagta ccccgtgttc
     2821 agaggcagac ccagcggcaa tgagagccag cacagactgg acttccagct gatgctgaag
     2881 atccgggaca ccctgtatat cgccggcaga gatcaggtgt acacagtgaa cctgaacgag
     2941 atgcccaaga ccgaagtgat ccccaacaag aagctgacct ggcggagcag acagcaggac
     3001 agagaaaact gcgccatgaa gggcaagcac aaggacgagt gccacaactt catcaaggtg
     3061 ttcgtgcccc ggaacgacga gatggtgttt gtgtgcggca ccaacgcctt caatcccatg
     3121 tgccggtact acagactgag caccctggaa tacgacggcg aggaaatcag cggcctggcc
     3181 agatgtcctt tcgacgccag acagaccaac gtggccctgt ttgccgatgg caagctgtac
     3241 tctgctaccg tggccgattt tctggccagc gacgctgtga tctaccggtc catgggagat
     3301 ggcagcgccc tgcggaccat taagtacgac agcaagtgga tcaaagagcc ccacttcctg
     3361 cacgccatcg agtacggcaa ctacgtgtac ttcttcttcc gggaaatcgc cgtggaacac
     3421 aacaacctgg gcaaagccgt gtacagcaga gtggcccgga tctgcaagaa cgatatgggc
     3481 ggctctcaga gagtgctgga aaagcactgg accagctttc tgaaggcccg gctgaattgc
     3541 agcgtgcccg gcgatagctt cttctacttc gacgtgctgc agagcatcac cgacatcatc
     3601 cagatcaacg gcatccccac agtcgtgggc gtgttcacca cacagctgaa cagcatccct
     3661 ggctctgccg tgtgcgcctt cagcatggac gatatcgaga aggtgttcaa aggccggttc
     3721 aaagagcaga aaacccctga cagcgtgtgg accgccgtgc ctgaagataa ggtgcccaag
     3781 cctagacctg gctgctgtgc caaacatgga ctggccgagg cctacaagac cagcatcgac
     3841 ttccccgacg agacactgag ctttatcaag tctcaccctc tgatggacag cgccgtgcca
     3901 cctattgccg acgagccctg gtttaccaag accagagtgc ggtacaggct gaccgccatc
     3961 tctgtggatc atagcgccgg accttaccag aactacaccg tgatcttcgt gggcagcgag
     4021 gccggaatgg tgctgaaagt gctggccaag acaagcccct tcagcctgaa cgattccgtg
     4081 ctgctggaag agatcgaggc ctataaccac gccaagtgca gcgccgagaa cgaagaggac
     4141 aagaaagtga tcagcctgca gctggacaag gaccatcacg ccctgtacgt ggccttcagc
     4201 tcctgtatca tcagaatccc tctgagccgc tgcgagagat acggcagctg caagaagtcc
     4261 tgtatcgcca gcagggaccc ttactgtggc tggctgtctc agggctcttg cggcagagtt
     4321 acacctggaa tgctggccga aggctacgag caggataccg agtttggcaa cacagcccac
     4381 ctgggcgact gccacgaaat cctgcctacc agcaccacac ctgactacaa gatcttcggc
     4441 ggacccacca gcgacatgga agtgtctagc agcagcgtga ccaccatggc ctccattcca
     4501 gagatcaccc ctaaagtgat cgacacctgg cggcccaagc tgaccagcag cagaaagttc
     4561 gtggtgcagg acgaccccaa cacctccgac tttaccgatc ctctgagcgg catccctaag
     4621 ggcgtcagat gggaagtgca gagcggcgag agcaaccaga tggtgcacat gaatggcgcg
     4681 ccgggcggcg acaagaccca cacctgcccc ccctgcccag ctccagaact gctgggaggg
     4741 ccttccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag caggaccccc
     4801 gaggtgacct gcgtggtggt ggccgtgtcc cacgaggacc cagaggtgaa gttcaactgg
     4861 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagagagga gcagtacaac
     4921 agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa
     4981 gaatacaagt gcaaagtctc caacaaggcc ctggctgccc caatcgaaaa gacaatcagc
     5041 aaggccaagg gccagccacg ggagccccag gtgtacaccc tgccccccag ccgggaggag
     5101 atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc cagcgatatc
     5161 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg
     5221 ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtccaggtgg
     5281 cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
     5341 cagaagtccc tgagcctgag ccccggcggc agcggtctta atgatatttt cgaggctcag
     5401 aagattgagt ggcatgaaca ccaccaccat caccactaag gccggcctgt agtcactggc
     5461 acaacgcgtt acccggtaag ccaatcgggt atacacggtc gtcatactgc agacagggtt
     5521 cttctacttt gcaagatagt cttgagtagt aaaataaata gatagagaaa atcagcctcg
     5581 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5641 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcacaacac
     5701 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5761 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     5821 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5881 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5941 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     6001 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     6061 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     6121 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     6181 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     6241 aaggtgagga actaaaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     6301 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     6361 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     6421 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     6481 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     6541 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     6601 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     6661 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     6721 ggggtgcccg ccttcctgga gacatccgcg ccccgcaacc tccccttcta cgagcggctc
     6781 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     6841 cgcaagcccg gtgcctgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa
     6901 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     6961 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     7021 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     7081 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     7141 agcttggcgt aatcatggtc attaccaatg cttaatcagt gaggcaccta tctcagcgat
     7201 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7261 ggagggctta ccatctggcc ccagcgctgc gatgataccg cgagaaccac gctcaccggc
     7321 tccggattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7381 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7441 gccagttaat agtttgcgca acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc
     7501 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7561 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7621 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7681 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7741 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7801 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7861 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7921 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7981 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atattcttcc tt
//
