LOCUS       sequence-72768-e        7012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Flag tagged mouse Tim3 (T1 truncation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7012)
  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 7012)
  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 (T1 truncation)
FEATURES             Location/Qualifiers
     source          1..7012
                     /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(2564..2582)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2565..2582)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2602..2619)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2608..2832
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2878..3306
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2965..2984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3175..3196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3315..3335)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3320..3650
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3501..3636
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3563..3582
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3717..4511
                     /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(3771..3790)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4381..4400
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4685..4806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4722..4741)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4776..4795
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4855..4871)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4855..4871)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4868..4890)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4879..4895
                     /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(4903..4909,4910..4927,4928..4933))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4948..4969
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5086..5103)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5257..5845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5337..5356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6016..6807,6808..6876))
                     /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     6639..6658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6877..6981)
                     /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 tgacagaatt ccagctgagc gccggtcgct accattacca gttggtctgg tgtcaaaaat
     2461 aataaccggg caggccatgt ctgcccgtat ttcgcgtaag gaaatccatt atgtactatt
     2521 taaactcgag cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa
     2581 atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2641 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2701 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2761 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2821 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2881 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2941 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3001 tcgccggctt tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg
     3061 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3121 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3181 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3241 ggattttggg gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3301 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg
     3361 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3421 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3481 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3541 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3601 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3661 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3721 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3781 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3841 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3901 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3961 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4021 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4081 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4141 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4201 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4261 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4321 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4381 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4441 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4501 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4561 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4621 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4681 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4741 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4801 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4861 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4921 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4981 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5041 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5101 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5161 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5221 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5281 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5341 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5401 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5461 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5521 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5581 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5641 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5701 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5761 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5821 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5881 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5941 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6001 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6061 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6121 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6181 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6241 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6301 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6361 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6421 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6481 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6541 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6601 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6661 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6721 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6781 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6841 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6901 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6961 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
