LOCUS       sequence-302559-        6958 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 6958)
  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 6958)
  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-MICA-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6958
                     /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             4300..4344
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4345..4362
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4502..4519)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4508..4619
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4737..4757)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4742..5071
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4922..5057
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4984..5003
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5119..5166
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5185..5784
                     /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(5185..5204)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5681..5701
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5914..6035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5951..5970)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6005..6024
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6088..6879,6880..6948))
                     /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     6711..6730
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6949..6958,1..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 cggccgccga acctcacagc
     2761 ctgagataca atctgaccgt gctgagctgg gatggcagcg tgcagtctgg atttctgacc
     2821 gaagtgcacc tggacggcca gcctttcctg agatgcgaca gacagaagtg cagagccaag
     2881 cctcaaggcc agtgggctga agatgtgctg ggcaacaaga cctgggacag agagacaaga
     2941 gatctgaccg gcaacggcaa ggacctgaga atgacactgg cccacatcaa ggaccagaaa
     3001 gagggcctgc acagcctgca agagatcaga gtgtgcgaga tccacgagga caacagcacc
     3061 agaagcagcc agcacttcta ctacgacggc gagctgttcc tgagccagaa cctggaaaca
     3121 aaagaatgga ccatgcctca gagcagcaga gcccagacac tggctatgaa cgtgcggaac
     3181 ttcctgaaag aggacgccat gaagaccaag acacactacc acgccatgca cgccgactgt
     3241 ctgcaagagc tgagaagata cctgaagtcc ggcgtggtgc tgagaaggac agtgcctcct
     3301 atggtcaacg tgaccagatc tgaggccagc gagggcaata tcaccgtgac atgtagagcc
     3361 agcggcttct acccctggaa catcaccctg tcttggagac aggatggcgt gtccctgagc
     3421 cacgatacac agcagtgggg agatgtcctg cctgacggca atggcacata ccagacatgg
     3481 gtcgccacca gaatctgcca gggcgaagaa cagcggttca cctgttacat ggaacacagc
     3541 ggcaaccaca gcacacaccc tgtgccatct ggaaaggtgc tggtgctgca gtctcactgg
     3601 ggcgcgccgg gcggcgacaa gacccacacc tgccccccct gcccagctcc agaactgctg
     3661 ggagggcctt ccgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcagg
     3721 acccccgagg tgacctgcgt ggtggtggcc gtgtcccacg aggacccaga ggtgaagttc
     3781 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag
     3841 tacaacagca cctacagggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac
     3901 ggcaaagaat acaagtgcaa agtctccaac aaggccctgg ctgccccaat cgaaaagaca
     3961 atcagcaagg ccaagggcca gccacgggag ccccaggtgt acaccctgcc ccccagccgg
     4021 gaggagatga ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctaccccagc
     4081 gatatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc
     4141 ccagtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagtcc
     4201 aggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac
     4261 tacacccaga agtccctgag cctgagcccc ggcggcagcg gtcttaatga tattttcgag
     4321 gctcagaaga ttgagtggca tgaacaccac caccatcacc actaaggccg gcctgtagtc
     4381 actggcacaa cgcgttaccc ggtaagccaa tcgggtatac acggtcgtca tactgcagac
     4441 agggttcttc tactttgcaa gatagtcttg agtagtaaaa taaatagata gagaaaatca
     4501 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4561 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatca
     4621 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4681 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     4741 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4801 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4861 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4921 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4981 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     5041 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     5101 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     5161 tacgacaagg tgaggaacta aaccatgacc gagtacaagc ccacggtgcg cctcgccacc
     5221 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     5281 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     5341 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     5401 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     5461 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     5521 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     5581 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     5641 cgcgccgggg tgcccgcctt cctggagaca tccgcgcccc gcaacctccc cttctacgag
     5701 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     5761 atgacccgca agcccggtgc ctgacacgtg ctacgagatt tcgattccac cgccgccttc
     5821 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5881 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5941 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     6001 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     6061 agctagagct tggcgtaatc atggtcatta ccaatgctta atcagtgagg cacctatctc
     6121 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6181 gatacgggag ggcttaccat ctggccccag cgctgcgatg ataccgcgag aaccacgctc
     6241 accggctccg gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6301 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6361 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc
     6421 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6481 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6541 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6601 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6661 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6721 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6781 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6841 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6901 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatat tcttcctt
//
