LOCUS       sequence-72767-e        6875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Flag tagged mouse Tim3 fragment (no cytosolic domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6875)
  AUTHORS   Lee J, Su EW, Zhu C, Hainline S, Phuah J, Moroco JA, Smithgall TE, 
            Kuchroo VK, Kane LP
  TITLE     Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor 
            signaling pathways.
  JOURNAL   Mol Cell Biol. 2011 Oct;31(19):3963-74. doi: 10.1128/MCB.05297-11. 
            Epub 2011 Aug 1.
  PUBMED    21807895
REFERENCE   2  (bases 1 to 6875)
  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: 
            "10.1128/MCB.05297-11"; journalName: "Mol Cell Biol"; date: 
            "2011-10"; volume: "31"; issue: "19"; pages: "3963-74"
COMMENT     Sequence Label: Flag‐mTim3 (del.Cyto)
FEATURES             Location/Qualifiers
     source          1..6875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        353..1531
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          584..1522
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1479..1499
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1624..1647
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2427..2445)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2428..2445)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2465..2482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2471..2695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2741..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3038..3059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3178..3198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3183..3513
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3364..3499
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3426..3445
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3580..4374
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3634..3653)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4244..4263
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4548..4669
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4585..4604)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4639..4658
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4718..4734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4718..4734)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4731..4753)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4742..4758
                     /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(4766..4772,4773..4790,4791..4796))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4811..4832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4949..4966)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5200..5219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5879..6670,6671..6739))
                     /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     6502..6521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6740..6844)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac taggcttttg caaaaagctt tgcaaagatg gataaagttt taaacagaga
      301 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgcctcgt gaggctccgg
      361 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      421 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      481 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      541 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      601 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      661 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      721 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      781 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      841 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
      901 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
      961 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1021 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1081 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1141 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1201 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1261 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1321 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1381 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1441 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1501 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtaccga gctcggatcc
     1561 atggccttac cagtgaccgc cttgctcctg ccgctagcct tgctgctcca cgccgccagg
     1621 ccggattaca aggacgatga cgacaagatc gatttggaaa atgcttatgt gtttgaggtt
     1681 ggtaagaatg cctatctgcc ctgcagttac actctatcta cacctggggc acttgtgcct
     1741 atgtgctggg gcaagggatt ctgtccttgg tcacagtgta ccaacgagtt gctcagaact
     1801 gatgaaagaa atgtgacata tcagaaatcc agcagatacc agctaaaggg cgatctcaac
     1861 aaaggagacg tgtctctgat cataaagaat gtgactctgg atgaccatgg gacctactgc
     1921 tgcaggatac agttccctgg tcttatgaat gataaaaaat tagaactgaa attagacatc
     1981 aaagcagcca aggtcactcc agctcagact gcccatgggg actctactac agcttctcca
     2041 agaaccctaa ccacggagag aaatggttca gagacacaga cactggtgac cctccataat
     2101 aacaatggaa caaaaatttc cacatgggct gatgaaatta aggactctgg agaaacgatc
     2161 agaactgcta tccacattgg agtgggagtc tctgctgggt tgaccctggc acttatcatt
     2221 ggtgtcttaa tccttaaatg gttttcctgt tgacagctga agggcgaatt ccagctgagc
     2281 gccggtcgct accattacca gttggtctgg tgtcaaaaat aataataacc gggcaggcca
     2341 tgtctgcccg tatttcgcgt aaggaaatcc attatgtact atttaaactc gagcggccgc
     2401 tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
     2461 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2521 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2581 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2641 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2701 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3061 acaacactca accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg
     3121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     3181 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa
     3241 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3301 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3361 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3421 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3481 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3541 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3601 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3661 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3721 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3781 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3841 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3901 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3961 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4021 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4081 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4141 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4201 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4261 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4321 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4381 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4441 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4501 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4561 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4621 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4681 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4741 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4801 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4921 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4981 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5041 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5221 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5281 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     5341 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5401 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5461 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5521 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5581 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5641 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6001 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6781 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6841 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
