LOCUS       sequence-72764-e        7045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Flag tagged mouse Tim3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7045)
  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 7045)
  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
FEATURES             Location/Qualifiers
     source          1..7045
                     /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(2597..2615)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2598..2615)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2635..2652)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2641..2865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2911..3339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2998..3017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3208..3229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3348..3368)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3353..3683
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3534..3669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3596..3615
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3750..4544
                     /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(3804..3823)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4414..4433
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4718..4839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4755..4774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4809..4828
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4888..4904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4888..4904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4901..4923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4912..4928
                     /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(4936..4942,4943..4960,4961..4966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4981..5002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5119..5136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5290..5878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5370..5389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6049..6840,6841..6909))
                     /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     6672..6691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6910..7014)
                     /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 atatctacac catcgaggag aacgtatatg aagtggagaa ttcaaatgag
     2401 tactactgct acgtcaacag ccagcagcca tcctgacaga attccagctg agcgccggtc
     2461 gctaccatta ccagttggtc tggtgtcaaa aataataacc gggcaggcca tgtctgcccg
     2521 tatttcgcgt aaggaaatcc attatgtact atttaaactc gagcggccgc tcgagcatgc
     2581 atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga
     2641 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2701 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2761 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2821 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     2881 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     2941 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3001 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3061 atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3121 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3181 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3241 accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt
     3301 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     3361 gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc
     3421 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3481 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3541 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3601 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3661 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3721 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3781 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3841 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3901 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3961 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4021 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4081 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4141 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4201 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4261 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4321 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4381 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4441 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4501 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4561 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4621 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4681 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4741 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4801 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4861 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4921 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4981 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5041 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5101 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5161 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5221 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5281 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5341 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5401 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5461 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     5521 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5581 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5641 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5701 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5761 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5821 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5881 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5941 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6001 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6061 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6121 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6181 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6241 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6301 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6361 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6421 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6481 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6541 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6601 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6661 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6721 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6781 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6841 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6901 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6961 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7021 tccccgaaaa gtgccacctg acgtc
//
