LOCUS       sequence-447063-        6820 bp    DNA     circular SYN 14-APR-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 6820)
  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 6820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-NCR3LG1-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..91
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     97..116
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      202..790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     691..710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     944..961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1059..1076
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1060..1076
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1532..1548
                     /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        1715..2094
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2095..2298
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2249..2269
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2295..2319
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2529..2547
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2529..2546
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2560..2579
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4110..4154
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4155..4172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4312..4329)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4318..4429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4547..4567)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4552..4881
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4732..4867
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4794..4813
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4929..4976
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4995..5594
                     /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(4995..5014)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5491..5511
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5724..5845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5761..5780)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5815..5834
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5898..6689,6690..6758))
                     /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     6521..6540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6759..6820,1..30))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tatttgaatg tatttagaaa aataaacaaa taggggtcag tgttacaacc aattaaccaa
       61 ttctgaacat tatcgcgagc ccatttatac ctgaatatgg ctcataacac cccttgctca
      121 tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt
      181 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      241 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      301 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      361 gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      421 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      481 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      541 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      601 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      661 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      721 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      781 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      841 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      901 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      961 ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac
     1021 ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta
     1081 agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa
     1141 aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaagagcatt tgtcagaata
     1201 tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa
     1261 aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta
     1321 aactattcat ctattattta tgattttttg tatatacaat atttctagtt tgttaaagag
     1381 aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat atcaaaatta
     1441 tacatgtcaa cgataataca aaatataata caaactataa gatgttatca gtatttatta
     1501 tcatttagaa taaattttgt gtcgccctta attgtgagcg gataacaatt acgagcttca
     1561 tgcacagtgg cgttgactca atattggcca ttagccatat tattcattgg ttatatagca
     1621 taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt
     1681 tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac tagttattaa
     1741 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     1801 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1861 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     1921 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     1981 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     2041 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     2101 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     2161 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     2221 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     2281 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
     2341 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg ggaacggtgc
     2401 attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg
     2461 cccaccccct tggcttcgtt agaacgcggc tacaattaat acataacctt atgtatcata
     2521 cacatacgat ttaggtgaca ctatagaata acatccactt tgcctttctc tccacaggtg
     2581 tccactccca ggtccaactg cacctcggtt ctatcgaaaa cgcgcctgct agcgccacca
     2641 tgaagaccat cattgccctg agctacatct tctgcctggt gttcgctgcg gccgccgatc
     2701 tgaaggttga aatgatggct ggcggcaccc agatcacccc tctgaacgac aatgtgacca
     2761 tcttctgcaa catcttctac agccagcctc tgaacatcac cagcatgggc atcacctggt
     2821 tctggaagtc cctgaccttc gacaaagaag tgaaggtttt cgagttcttc ggcgaccacc
     2881 aagaggcctt tagacctggc gctatcgtgt ccccttggag actgaaaagc ggcgacgctt
     2941 ctctgagact gcccggcatt caactggaag aggccggcga gtacagatgc gaggttgtgg
     3001 ttacacctct gaaggcccag ggcacagtgc agctggaagt tgtggcttct ccagcctcca
     3061 gactgctgct ggatcaagtg ggcatgaagg aaaacgagga caagtacatg tgcgagagca
     3121 gcggcttcta ccccgaggcc atcaatatca cctgggagaa gcagacccag aagttccctc
     3181 atcctatcga gatcagcgag gacgtgatca caggccccac catcaagaac atggacggca
     3241 ccttcaacgt gaccagctgc ctgaagctga acagctctca agaggaccct ggcaccgtgt
     3301 atcagtgtgt tgtcagacac gccagcctgc acaccccact gagaagcaat ttcaccctga
     3361 cagccgccag acacagcctg agcgagacag agaaaaccga caacttctct ggcgcgccgg
     3421 gcggcgacaa gacccacacc tgccccccct gcccagctcc agaactgctg ggagggcctt
     3481 ccgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcagg acccccgagg
     3541 tgacctgcgt ggtggtggcc gtgtcccacg aggacccaga ggtgaagttc aactggtacg
     3601 tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag tacaacagca
     3661 cctacagggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat
     3721 acaagtgcaa agtctccaac aaggccctgg ctgccccaat cgaaaagaca atcagcaagg
     3781 ccaagggcca gccacgggag ccccaggtgt acaccctgcc ccccagccgg gaggagatga
     3841 ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctaccccagc gatatcgccg
     3901 tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc ccagtgctgg
     3961 acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc aggtggcagc
     4021 agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga
     4081 agtccctgag cctgagcccc ggcggcagcg gtcttaatga tattttcgag gctcagaaga
     4141 ttgagtggca tgaacaccac caccatcacc actaaggccg gcctgtagtc actggcacaa
     4201 cgcgttaccc ggtaagccaa tcgggtatac acggtcgtca tactgcagac agggttcttc
     4261 tactttgcaa gatagtcttg agtagtaaaa taaatagata gagaaaatca gcctcgactg
     4321 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4381 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatca caacactcaa
     4441 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     4561 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4621 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4681 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4741 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4801 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4861 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4921 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     4981 tgaggaacta aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     5041 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5101 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5161 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5221 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5281 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5341 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     5401 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5461 tgcccgcctt cctggagaca tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5521 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5581 agcccggtgc ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     5641 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5701 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5761 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5821 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5881 tggcgtaatc atggtcatta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5941 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6001 ggcttaccat ctggccccag cgctgcgatg ataccgcgag aaccacgctc accggctccg
     6061 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6121 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6181 gttaatagtt tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg
     6241 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6301 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6361 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6421 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6481 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6541 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6601 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6661 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6721 aagggaataa gggcgacacg gaaatgttga atactcatat tcttcctttt tcaatattat
     6781 tgaagcattt atcagggtta ttgtctcatg agcggataca
//
