LOCUS       sequence-72769-e        6970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Flag tagged mouse Tim3 (T2 truncation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  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 6970)
  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 (T2 truncation)
FEATURES             Location/Qualifiers
     source          1..6970
                     /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(2522..2540)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2523..2540)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2560..2577)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2566..2790
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2836..3264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2923..2942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3133..3154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3273..3293)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3278..3608
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3459..3594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3521..3540
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3675..4469
                     /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(3729..3748)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4339..4358
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4643..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4680..4699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4734..4753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4813..4829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4813..4829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4826..4848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4837..4853
                     /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(4861..4867,4868..4885,4886..4891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4906..4927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5044..5061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5215..5803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5295..5314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5974..6765,6766..6834))
                     /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     6597..6616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6835..6939)
                     /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 gtattcctgt aagaaaaaga agttatcgag tttgagcctt
     2281 attacactgg ccaacttgcc tccaggaggg ttggcaaatg caggagcagt caggattcgc
     2341 tctgaggaaa atatctgaca gaattccagc tgagcgccgg tcgctaccat taccagttgg
     2401 tctggtgtca aaaataataa taaccgggca ggccatgtct gcccgtattt cgcgtaagga
     2461 aatccattat gtactattta aactcgagcg gccgctcgag catgcatcta gagggcccta
     2521 ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt
     2581 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2641 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2701 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2761 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2821 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2881 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2941 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt
     3001 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3061 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3121 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3181 attcttttga tttataaggg attttgggga tttcggccta ttggttaaaa aatgagctga
     3241 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3301 gtccccaggc tccccaggca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3361 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3421 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3481 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3541 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3601 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3661 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3721 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3781 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3841 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3901 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3961 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4021 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4081 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4141 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4201 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4261 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4321 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4381 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4441 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4501 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4561 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4621 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4681 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4741 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4801 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4861 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4921 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4981 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5041 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5101 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5161 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5221 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5281 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5341 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5401 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5461 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5521 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5581 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5641 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5701 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5761 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5821 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5881 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5941 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6001 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6061 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6121 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6181 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6241 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6301 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6361 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6421 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6481 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6541 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6601 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6661 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6721 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6781 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6841 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6901 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6961 acctgacgtc
//
