LOCUS       sequence-368822-        6466 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 6466)
  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 6466)
  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-TIGIT-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6466
                     /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(2188..2207)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(2220..2238)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2221..2238)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2448..2472)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2469..2672)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2498..2518)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2673..3052
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    3219..3235
                     /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(3691..3708)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3691..3707)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3806..3823)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3977..4565)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4057..4076)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4651..4670)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4676..4695)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4737..4828
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4829..4897,4898..5689)
                     /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(5047..5066)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    5742..5863
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5753..5772)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5807..5826
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5993..6466,1..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(6076..6096)
                     /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 aggaatctga aatctggcgc catgctcggc
     1741 cacgctgctt tccagcactt ccagaaagat tctgccggtg taggtgccgt cggggtatgt
     1801 gtggtagatg cagaagtact cgccggtatc gttcacggtc aggctctgaa gtgtcagtcc
     1861 caggccaggt ccaggagcca ctctatcctt gaagctgggg ctgatgtgcc atccgagatc
     1921 ggcattacag atggccagca gctgatcctg ctgctcccag ttcacttggg tcacttgagc
     1981 ggtggtgcta gacaggtgac actgcaggat gatgctgccg cctttctcgg cagagatgtt
     2041 gccggttgtc tcgattgtgc ctgtcatcat ggcggccgca gcgaacacca ggcagaagat
     2101 gtagctcagg gcaatgatgg tcttcatggt ggcgctagca ggcgcgtttt cgatagaacc
     2161 gaggtgcagt tggacctggg agtggacacc tgtggagaga aaggcaaagt ggatgttatt
     2221 ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg
     2281 cgttctaacg aagccaaggg ggtgggccta tagactctat aggcggtact tacgtcactc
     2341 ttggcacggg gaatccgcgt tccaatgcac cgttcccggc cgcggaggct ggatcggtcc
     2401 cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc
     2461 actaaacgag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     2521 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     2581 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     2641 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     2701 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     2761 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     2821 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     2881 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     2941 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     3001 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg ccaacatggc
     3061 ggtcatattg gacatgagcc aatataaatg tacatattat gatatagata caacgtatgc
     3121 aatggccaat agccaatatt gatttatgct atataaccaa tgaataatat ggctaatggc
     3181 caatattgag tcaacgccac tgtgcatgaa gctcgtaatt gttatccgct cacaattaag
     3241 ggcgacacaa aatttattct aaatgataat aaatactgat aacatcttat agtttgtatt
     3301 atattttgta ttatcgttga catgtataat tttgatatca aaaactgatt ttccctttat
     3361 tattttcgag atttattttc ttaattctct ttaacaaact agaaatattg tatatacaaa
     3421 aaatcataaa taatagatga atagtttaat tataggtgtt catcaatcga aaaagcaacg
     3481 tatcttattt aaagtgcgtt gcttttttct catttataag gttaaataat tctcatatat
     3541 caagcaaagt gacaggcgcc cttaaatatt ctgacaaatg ctctttccct aaactccccc
     3601 cataaaaaaa cccgccgaag cgggttttta cgttatttgc ggattaacga ttactcgtta
     3661 tcagaaccgc ccagggggcc cgagcttaag actggccgtc gttttacaac acagaaagag
     3721 tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct gcttagtttg atgcctggca
     3781 gttccctact ctcgccttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3841 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3901 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3961 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4021 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4081 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4141 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4201 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4261 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4321 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4381 tcttgaagtg gtgggctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4441 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4501 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4561 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gacgcgcgcg
     4621 taactcacgt taagggattt tggtcatgag caaggggtgt tatgagccat attcaggtat
     4681 aaatgggctc gcgataatgt tcagaattgg ttaattggtt gtaacactga cccctatttg
     4741 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4801 gcttcaataa tattgaaaaa ggaagaatat gagtattcaa catttccgtg tcgcccttat
     4861 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4921 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4981 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5041 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5101 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5161 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5221 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5281 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5341 accaaacgac gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt tgcgcaaact
     5401 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5461 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5521 taaatccgga gccggtgagc gtggttctcg cggtatcatc gcagcgctgg ggccagatgg
     5581 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5641 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaat gaccatgatt
     5701 acgccaagct ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt
     5761 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     5821 tattgcttta tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact
     5881 ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga
     5941 agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg tgtcaggcac
     6001 cgggcttgcg ggtcatgcac caggtgcgcg gtccttcggg cacctcgacg tcggcggtga
     6061 cggtgaagcc gagccgctcg tagaagggga ggttgcgggg cgcggatgtc tccaggaagg
     6121 cgggcacccc ggcgcgctcg gccgcctcca ctccggggag cacgacggcg ctgcccagac
     6181 ccttgccctg gtggtcgggc gagacgccga cggtggccag gaaccacgcg ggctccttgg
     6241 gccggtgcgg cgccaggagg ccttccatct gttgctgcgc ggccagccgg gaaccgctca
     6301 actcggccat gcgcgggccg atctcggcga acaccgcccc cgcttcgacg ctctccggcg
     6361 tggtccagac cgccaccgcg gcgccgtcgt ccgcgaccca caccttgccg atgtcgagcc
     6421 cgacgcgcgt gaggaagagt tcttgcagct cggtgacccg ctcgat
//
