LOCUS       sequence-235998-        8283 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expression of human PD-L1 mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8283)
  AUTHORS   Li CW, Lim SO, Xia W, Lee HH, Chan LC, Kuo CW, Khoo KH, Chang SS, 
            Cha JH, Kim T, Hsu JL, Wu Y, Hsu JM, Yamaguchi H, Ding Q, Wang Y, 
            Yao J, Lee CC, Wu HJ, Sahin AA, Allison JP, Yu D, Hortobagyi GN, 
            Hung MC
  TITLE     Glycosylation and stabilization of programmed death ligand-1 
            suppresses T-cell activity.
  JOURNAL   Nat Commun. 2016 Aug 30;7:12632. doi: 10.1038/ncomms12632.
  PUBMED    27572267
REFERENCE   2  (bases 1 to 8283)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2016-08-30"; volume: "7:12632. doi"; pages: " 
            10.1038/ncomms12632"
COMMENT     Sequence Label: pCDH-puro/Flag PD-L1 N200Q/N219Q
FEATURES             Location/Qualifiers
     source          1..8283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        83..286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     237..257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     283..307
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1239..1262
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1307..1518
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1531..1799
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             1826..2425
                     /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(1826..1845)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2322..2342
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2432..3020
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2485..2505)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2903..2914)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3094..3327
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3399..3520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3436..3455)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3490..3509
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3560..3695
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3728..3744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3728..3744)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3741..3763)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3752..3768
                     /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)."
     promoter        complement(join(3776..3782,3783..3800,3801..3806))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3821..3842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3959..3976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4130..4718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4889..5680,5681..5749))
                     /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     5512..5531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5750..5854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5922..5940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5978..6000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6100..6119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6313..6335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6327..6344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6328..6344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6359..6585
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6586..6766
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6813..6938
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7431..7664
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7849..7893
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8160..8276
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ctagtattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
       61 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      121 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      181 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      241 aatgggcggt aggcgtgtac ggtgggaggt ttatataagc agagctcgtt tagtgaaccg
      301 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagat tctagagcta
      361 gcgaattcat gaggatattt gctgtcttta tattcatgac ctactggcat ttgctgaacg
      421 catttactgt cacggttccc aaggacctat atgtggtaga gtatggtagc aatatgacaa
      481 ttgaatgcaa attcccagta gaaaaacaat tagacctggc tgcactaatt gtctattggg
      541 aaatggagga taagaacatt attcaatttg tgcatggaga ggaagacctg aaggttcagc
      601 atagtagcta cagacagagg gcccggctgt tgaaggacca gctctccctg ggaaatgctg
      661 cacttcagat cacagatgtg aaattgcagg atgcaggggt gtaccgctgc atgatcagct
      721 atggtggtgc cgactacaag cgaattactg tgaaagtcaa tgccccatac aacaaaatca
      781 accaaagaat tttggttgtg gatccagtca cctctgaaca tgaactgaca tgtcaggctg
      841 agggctaccc caaggccgaa gtcatctgga caagcagtga ccatcaagtc ctgagtggta
      901 agaccaccac caccaattcc aagagagagg agaagctttt caatgtgacc agcacactga
      961 gaatccagac aacaactaat gagattttct actgcacttt taggagatta gatcctgagg
     1021 aacagcatac agctgaattg gtcatcccag aactacctct ggcacatcct ccaaatgaaa
     1081 ggactcactt ggtaattctg ggagccatct tattatgcct tggtgtagca ctgacattca
     1141 tcttccgttt aagaaaaggg agaatgatgg atgtgaaaaa atgtggcatc caagatacaa
     1201 actcaaagaa gcaaagtgat acacatttgg aggagacgga ctacaaagac gatgacgaca
     1261 agtgagcggc cgcgaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     1321 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     1381 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     1441 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     1501 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     1561 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     1621 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     1681 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     1741 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
     1801 aagctgtgac cggcgcctac gctagatgac cgagtacaag cccacggtgc gcctcgccac
     1861 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     1921 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     1981 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     2041 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2101 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     2161 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     2221 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     2281 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     2341 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     2401 catgacccgc aagcccggtg cctgagtcga caatcaacct ctggattaca aaatttgtga
     2461 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     2521 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     2581 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     2641 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     2701 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     2761 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     2821 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     2881 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     2941 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3001 cctttgggcc gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag
     3061 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     3121 gataagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     3181 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     3241 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     3301 tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt
     3361 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
     3421 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3481 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat
     3541 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3601 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3661 attccagaag tagtgaggag gcttttttgg aggcctagac ttttgcagag accaaattcg
     3721 taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3781 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3841 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3901 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3961 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4561 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4861 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4921 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4981 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5041 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5101 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5161 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5221 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5281 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5341 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5401 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5461 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5521 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5581 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5641 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5701 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5761 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5821 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5881 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5941 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     6001 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     6061 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     6121 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     6181 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     6241 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     6301 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctgacgcgtg
     6361 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     6421 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     6481 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     6541 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     6601 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     6661 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     6721 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     6781 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6841 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6901 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6961 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     7021 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     7081 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     7141 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     7201 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     7261 ccaccgcaca gcaagcggcc actgatcttc agacctggag gaggagatat gagggacaat
     7321 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     7381 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     7441 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     7501 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     7561 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     7621 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     7681 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     7741 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     7801 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     7861 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     7921 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     7981 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     8041 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     8101 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcggttaact
     8161 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     8221 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaattcaa aattttatcg
     8281 ata
//
