LOCUS       sequence-393026-        6832 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 6832)
  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 6832)
  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-CD33-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6832
                     /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             4174..4218
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4219..4236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4376..4393)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4382..4493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4611..4631)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4616..4945
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4796..4931
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4858..4877
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4993..5040
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5059..5658
                     /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(5059..5078)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5555..5575
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5788..5909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5825..5844)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5879..5898
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5962..6753,6754..6822))
                     /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     6585..6604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6823..6832,1..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 tcctaatttc
     2761 tggctgcaag tgcaagagtc cgtgaccgtg caagagggac tgtgtgtgct ggtgccctgc
     2821 accttctttc accccattcc ttactacgac aagaacagcc ctgtgcacgg ctactggttt
     2881 agagagggcg ccatcatcag cagagatagc cctgtggcca ccaacaagct ggaccaagag
     2941 gtgcaagaag agacacaggg cagattcaga ctgctgggcg accccagcag aaacaactgt
     3001 agcctgagca tcgtggacgc caggcggaga gataacggca gctacttctt ccggatggaa
     3061 cggggcagca ccaagtacag ctacaagagc cctcagctgt ccgtgcacgt gaccgatctg
     3121 acccaccggc ctaagatcct gattcctggc acactggaac ccggccacag caagaatctg
     3181 acctgttctg tgtcttgggc ctgcgagcag ggcacacctc ctatcttttc ttggctgtcc
     3241 gccgctccta cctctctggg acctagaaca acacacagca gcgtgctgat catcacccct
     3301 agacctcagg accacggcac caacctgacc tgccaagtga aatttgctgg cgctggcgtg
     3361 accaccgaga gaaccattca gctcaacgtg acctacgtgc cacagaaccc taccaccgga
     3421 atctttccag gcgacggcag cggcaagcaa gaaacaagag ctggcgtggt gcatggcgcg
     3481 ccgggcggcg acaagaccca cacctgcccc ccctgcccag ctccagaact gctgggaggg
     3541 ccttccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag caggaccccc
     3601 gaggtgacct gcgtggtggt ggccgtgtcc cacgaggacc cagaggtgaa gttcaactgg
     3661 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagagagga gcagtacaac
     3721 agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa
     3781 gaatacaagt gcaaagtctc caacaaggcc ctggctgccc caatcgaaaa gacaatcagc
     3841 aaggccaagg gccagccacg ggagccccag gtgtacaccc tgccccccag ccgggaggag
     3901 atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc cagcgatatc
     3961 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg
     4021 ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtccaggtgg
     4081 cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
     4141 cagaagtccc tgagcctgag ccccggcggc agcggtctta atgatatttt cgaggctcag
     4201 aagattgagt ggcatgaaca ccaccaccat caccactaag gccggcctgt agtcactggc
     4261 acaacgcgtt acccggtaag ccaatcgggt atacacggtc gtcatactgc agacagggtt
     4321 cttctacttt gcaagatagt cttgagtagt aaaataaata gatagagaaa atcagcctcg
     4381 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4441 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcacaacac
     4501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     4621 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4681 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4741 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4801 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4861 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     4921 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     4981 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     5041 aaggtgagga actaaaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     5101 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     5161 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     5221 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     5281 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     5341 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     5401 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     5461 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     5521 ggggtgcccg ccttcctgga gacatccgcg ccccgcaacc tccccttcta cgagcggctc
     5581 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     5641 cgcaagcccg gtgcctgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa
     5701 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5761 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5821 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5881 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5941 agcttggcgt aatcatggtc attaccaatg cttaatcagt gaggcaccta tctcagcgat
     6001 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6061 ggagggctta ccatctggcc ccagcgctgc gatgataccg cgagaaccac gctcaccggc
     6121 tccggattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6181 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6241 gccagttaat agtttgcgca acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc
     6301 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6361 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6421 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6481 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6541 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6601 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6661 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6721 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6781 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atattcttcc tt
//
