LOCUS       sequence-462832-        6644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human GP78 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6644)
  AUTHORS   Zhang Z, Ren C, Xiao R, Ma S, Liu H, Dou Y, Fan Y, Wang S, Zhan P, 
            Gao C, Yue X, Li C, Gao L, Liang X, Wu Z, Ma C
  TITLE     Palmitoylation of TIM-3 promotes immune exhaustion and restrains 
            antitumor immunity.
  JOURNAL   Sci Immunol. 2024 Nov 15;9(101):eadp7302. doi: 
            10.1126/sciimmunol.adp7302. Epub 2024 Nov 15.
  PUBMED    39546589
REFERENCE   2  (bases 1 to 6644)
  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"; journalName: "Sci 
            Immunol."; date: "2024-11-15"; volume: "9(101):eadp7302. doi"; 
            pages: " 10.1126/sciimmunol.adp7302. Epub 2024 Nov 15"
COMMENT     Sequence Label: Flag-GP78
FEATURES             Location/Qualifiers
     source          1..6644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             931..996
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2985..3607
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3184..3203)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3631..3651)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3636..3965
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3816..3951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4003..4022)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4004..4022)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4036..4052)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4036..4052)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4049..4071)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4060..4076
                     /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(4084..4090,4091..4108,4109..4114))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4129..4150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4267..4284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4438..5026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4518..4537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5197..5988,5989..6057))
                     /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     5820..5839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6058..6162)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6189..6617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6276..6295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6486..6507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagcttg cggccgcgaa ttcaatgccg
     1021 ctgctcttcc tcgagcgctt cccctggccc agcctccgca cctacacggg cctcagcggc
     1081 ctggccctgc tgggcaccat catcagcgcc taccgcgcgc tcagccagcc cgaggccggc
     1141 cccggcgagc cggaccagct aacggcctcg ctgcagcctg agccgccggc gcccgcccgg
     1201 ccgagcgccg ggggaccccg ggcccgcgat gtggcccagt acctgctctc agacagcctc
     1261 ttcgtgtggg ttctagtaaa taccgcttgc tgtgttttga tgttggtggc taagctcatc
     1321 cagtgtattg tgtttggccc tcttcgagtg agtgagagac agcatctcaa agacaaattt
     1381 tggaatttta ttttctacaa gttcattttc atctttggtg tgctgaatgt ccagacagtg
     1441 gaagaggtgg tcatgtggtg cctctggttt gccggacttg tctttctgca cctgatggtt
     1501 cagctctgca aggatcgatt tgaatatctt tccttctcgc ccaccacgcc gatgagcagc
     1561 cacggtcgag tcctgtccct gttggttgcc atgctgcttt cctgctgtgg actggcggcc
     1621 gtctgctcca tcaccggcta cacccacgga atgcacacct tggctttcat ggctgcagag
     1681 tctcttcttg tgacagtgag gactgctcat gtgattttac gatacgtaat tcacctctgg
     1741 gacctcaacc acgaagggac gtgggaagga aaggggacgt atgtctatta cacagacttt
     1801 gtcatggagc tcactctcct gtccctggac ctcatgcacc atattcacat gttgttattt
     1861 ggcaacatct ggttatccat ggccagcctg gtcatcttta tgcagctgcg ttacctgttt
     1921 catgaggtgc aacgtcgaat tcgtcggcac aagaactatc tacgtgtggt tggaaacatg
     1981 gaggccaggt ttgcagttgc aactccagag gagctggctg tcaacaatga cgactgtgcc
     2041 atctgttggg actccatgca ggctgcgcgg aaactgccct gtggacatct tttccacaac
     2101 tcctgtcttc gttcctggct agaacaagac acctcctgtc caacatgcag aatgtctctt
     2161 aatattgccg acaataatcg tgtcagggaa gaacatcaag gagagaactt ggatgagaat
     2221 ttggttcctg tagcagcagc cgaagggaga cctcgcttaa accaacacaa tcacttcttc
     2281 catttcgatg ggtctcggat tgcgagctgg ctgccgagtt tttcggttga agtgatgcac
     2341 accaccaaca ttcttggcat tacgcaggcc agcaactccc agctcaatgc aatggctcat
     2401 cagattcaag agatgtttcc ccaggttcca taccatctgg tactgcagga cctccagctg
     2461 acacgctcag ttgaaataac aacagacaat attttagaag gacggattca agtacctttt
     2521 cctacacagc ggtcagatag catcagacct gcattgaaca gtcctgtgga aaggccaagc
     2581 agtgaccagg aagagggaga aacttctgct cagaccgagc gtgtgccact ggacctcagt
     2641 cctcgcctgg aggagacgct ggacttcggc gaggtggaag tggagcccag tgaggtggaa
     2701 gacttcgagg ctcgtgggag ccgcttctcc aagtctgctg atgagagaca gcgcatgctg
     2761 gtgcagcgta aggacgaact cctccagcaa gctcgcaaac gtttcttgaa caaaagttct
     2821 gaagatgatg cggcctcaga gagcttcctc ccctcggaag gtgcgtcctc tgaccccgtg
     2881 accctgcgtc gaaggatgct ggctgccgcc gcggaacgga ggcttcagaa gcagcagacc
     2941 tcctagagat ctgatatcgg taccagtcga ctctagagga tcccgggtgg catccctgtg
     3001 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     3061 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     3121 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     3181 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     3241 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     3301 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     3361 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     3421 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgatttta aaataactat
     3481 accagcagga ggacgtccag acacagcata ggctacctgg ccatgcccaa ccggtgggac
     3541 atttgagttg cttgcttggc actgtcctct catgcgttgg gtccactcag tagatgcctg
     3601 ttgaattggg tacgcggcca gcttggctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3661 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3721 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3901 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3961 gcaaaaagct cctcgaggaa ctgaaaaacc agaaagttaa ttccctatag tgagtcgtat
     4021 taaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4081 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4141 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4201 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4261 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4321 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4381 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4441 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4501 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4561 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4621 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4681 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4741 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4801 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4861 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4921 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4981 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5041 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5101 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5161 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5221 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5281 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5341 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5401 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5461 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5521 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5581 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5641 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5701 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5761 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5821 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5881 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5941 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6001 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6061 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6121 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6181 gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     6241 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     6301 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     6361 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     6421 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6481 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     6541 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     6601 aaaatttaac gcgaatttta acaaaatatt aacgcttaca attt
//
