LOCUS       sequence-368837-        6484 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 6484)
  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 6484)
  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-CTLA4-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..126
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(145..192)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(240..569)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      254..389
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(308..327)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     554..574
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    692..803
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     792..809
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(949..966)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(967..1011)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2206..2225)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2238..2256)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2239..2256)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2466..2490)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2487..2690)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2516..2536)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2691..3070
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    3237..3253
                     /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)."
     primer_bind     complement(3709..3726)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3709..3725)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3824..3841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3995..4583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4075..4094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4669..4688)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4694..4713)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4755..4846
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4847..4915,4916..5707)
                     /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     complement(5065..5084)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    5760..5881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5771..5790)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5825..5844
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6011..6484,1..126))
                     /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(6094..6114)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag
       61 ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc
      121 ggtcatggtt tagttcctca ccttgtcgta ttatactatg ccgatatact atgccgatga
      181 ttaattgtca acacgtgctg atcagatccg aaaatggata tacaagctcc cgggagcttt
      241 ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggca
      301 gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg
      361 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct
      421 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
      481 cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
      541 ggggactttc cacaccctaa ctgacacaca ttccacagaa ttaattcgcg ttaaattttt
      601 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
      661 aagaatagac cgagataggg ttgagtgttg tgatgcaatt tcctcatttt attaggaaag
      721 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga
      781 tggctggcaa ctagaaggca cagtcgaggc tgattttctc tatctattta ttttactact
      841 caagactatc ttgcaaagta gaagaaccct gtctgcagta tgacgaccgt gtatacccga
      901 ttggcttacc gggtaacgcg ttgtgccagt gactacaggc cggccttagt ggtgatggtg
      961 gtggtgttca tgccactcaa tcttctgagc ctcgaaaata tcattaagac cgctgccgcc
     1021 ggggctcagg ctcagggact tctgggtgta gtggttgtgc agggcctcgt gcatcacgct
     1081 gcagctgaac acgttgccct gctgccacct ggacttgtcc acggtcagct tgctgtacag
     1141 gaagaagctg ccgtcgctgt ccagcactgg gggggtggtc ttgtagttgt tctcgggctg
     1201 gccgttgctc tcccactcca cggcgatatc gctggggtag aagcccttca ccagacaggt
     1261 cagggacacc tggttcttgg tcatctcctc ccggctgggg ggcagggtgt acacctgggg
     1321 ctcccgtggc tggcccttgg ccttgctgat tgtcttttcg attggggcag ccagggcctt
     1381 gttggagact ttgcacttgt attctttgcc gttcagccag tcctggtgca gcacggtcag
     1441 cacggacacc accctgtagg tgctgttgta ctgctcctct ctgggcttgg tcttggcgtt
     1501 gtgcacctcc acgccgtcca cgtaccagtt gaacttcacc tctgggtcct cgtgggacac
     1561 ggccaccacc acgcaggtca cctcgggggt cctgctgatc atcagggtgt ccttgggctt
     1621 gggggggaac aggaacacgg aaggccctcc cagcagttct ggagctgggc agggggggca
     1681 ggtgtgggtc ttgtcgccgc ccggcgcgcc atcggagtca ggacaaggct cagggtcgat
     1741 cacgtagatc tgggtgccgt tgccgatgcc gaggtagtaa ggaggagggt acatcagttc
     1801 caccttgcag atatacaggc cggtgtccat ggctctcagg ccctggattg tcaggttcac
     1861 ttgattgccg ctgctggtgc ctgtacagat gctgtcgtcc aggaaggtca gctcattgcc
     1921 catcatgtag gtggcggcgc acacttctgt cacttggcta tcggcctgtc tcagcactgt
     1981 cacgcgcact tcagtggcct ttccaggaga ggcgtactcg cacacaaagc tggcgattcc
     2041 tctgctagag gccagcacca cagcaggctg agcaacatgc atagctttgg cggccgcagc
     2101 gaacaccagg cagaagatgt agctcagggc aatgatggtc ttcatggtgg cgctagcagg
     2161 cgcgttttcg atagaaccga ggtgcagttg gacctgggag tggacacctg tggagagaaa
     2221 ggcaaagtgg atgttattct atagtgtcac ctaaatcgta tgtgtatgat acataaggtt
     2281 atgtattaat tgtagccgcg ttctaacgaa gccaaggggg tgggcctata gactctatag
     2341 gcggtactta cgtcactctt ggcacgggga atccgcgttc caatgcaccg ttcccggccg
     2401 cggaggctgg atcggtcccg gtgtcttcta tggaggtcaa aacagcgtgg atggcgtctc
     2461 caggcgatct gacggttcac taaacgagct ctgcttatat agacctccca ccgtacacgc
     2521 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     2581 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     2641 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     2701 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     2761 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     2821 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     2881 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     2941 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     3001 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     3061 aatcaatgcc aacatggcgg tcatattgga catgagccaa tataaatgta catattatga
     3121 tatagataca acgtatgcaa tggccaatag ccaatattga tttatgctat ataaccaatg
     3181 aataatatgg ctaatggcca atattgagtc aacgccactg tgcatgaagc tcgtaattgt
     3241 tatccgctca caattaaggg cgacacaaaa tttattctaa atgataataa atactgataa
     3301 catcttatag tttgtattat attttgtatt atcgttgaca tgtataattt tgatatcaaa
     3361 aactgatttt ccctttatta ttttcgagat ttattttctt aattctcttt aacaaactag
     3421 aaatattgta tatacaaaaa atcataaata atagatgaat agtttaatta taggtgttca
     3481 tcaatcgaaa aagcaacgta tcttatttaa agtgcgttgc ttttttctca tttataaggt
     3541 taaataattc tcatatatca agcaaagtga caggcgccct taaatattct gacaaatgct
     3601 ctttccctaa actcccccca taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg
     3661 attaacgatt actcgttatc agaaccgccc agggggcccg agcttaagac tggccgtcgt
     3721 tttacaacac agaaagagtt tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc
     3781 ttagtttgat gcctggcagt tccctactct cgccttccgc ttcctcgctc actgactcgc
     3841 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3901 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3961 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4021 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4081 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4141 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4201 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4261 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4321 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4381 taggcggtgc tacagagttc ttgaagtggt gggctaacta cggctacact agaagaacag
     4441 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4501 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4561 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4621 agtggaacga cgcgcgcgta actcacgtta agggattttg gtcatgagca aggggtgtta
     4681 tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt
     4741 aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4801 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca
     4861 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4921 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4981 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5041 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5101 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5161 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5221 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5281 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5341 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc
     5401 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5461 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5521 tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc
     5581 agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5641 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5701 ttggtaatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     5761 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     5821 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     5881 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     5941 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     6001 gtagcacgtg tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca
     6061 cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg
     6121 cggatgtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca
     6181 cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga
     6241 accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg
     6301 ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg
     6361 cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca
     6421 ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct
     6481 cgat
//
