LOCUS       sequence-234346-        9035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human protein Gli containing a 4HT-inducible intein 
            evolved to operate at 37C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9035)
  AUTHORS   Peck SH, Chen I, Liu DR
  TITLE     Directed evolution of a small-molecule-triggered intein with 
            improved splicing properties in mammalian cells.
  JOURNAL   Chem Biol. 2011 May 27;18(5):619-30. doi: 
            10.1016/j.chembiol.2011.02.014.
  PUBMED    21609843
REFERENCE   2  (bases 1 to 9035)
  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: 
            "10.1016/j.chembiol.2011.02"; journalName: "Chem Biol"; date: 
            "2011-05-27- 27"; volume: "18"; issue: "5"; pages: "619-30"
COMMENT     Sequence Label: p3x-Flag Gli3 37R3-2
FEATURES             Location/Qualifiers
     source          1..9035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        147..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        527..730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     681..701
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4244..4309
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4320..4942
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4519..4538)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4966..4986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4984..5281
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5335..6129
                     /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(5389..5408)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5999..6018
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6658..7246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6738..6757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7417..8208,8209..8277))
                     /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     8040..8059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8278..8382)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8409..8864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8496..8515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8706..8727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8990..9012
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9004..9021
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9005..9021
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tacattgaat caatattggc aattagccat attagtcatt ggttatatag cataaatcaa
       61 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg
      121 ctcatgtcca atatgaccgc catgttgaca ttgattattg actagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtccgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tacgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      541 gcagtacacc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      601 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      661 taataacccc gccccgttga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      721 aagcagagct cgtttagtga accgtcagaa ttaagcttgc ggccgcacca tggaggccca
      781 ggcccacagc tctacggcga ctgagaggaa gaaagctgaa aattccattg ggaaatgtcc
      841 cacgagaaca gatgtcagcg agaaggccgt ggcctctagt accacttcca atgaggatga
      901 aagtcctgga cagatctatc accgagagag aagaaacgca atcactatgc agcctcagag
      961 tgtgcagggt ctcaacaaaa tcagtgagga gccctcgacg tctagtgatg agagggcctc
     1021 gctgatcaag aaagagatcc atggctctct accacatctg gcggagccct ctctccctta
     1081 ccgtgggact gtgtttgcca tggatccccg gaatggctac atggagcctc actaccaccc
     1141 tcctcatctt ttccctgcct tccatcctcc tgtaccaatt gatgccagac atcatgaggg
     1201 ccgttaccat tatgatccat ctcctattcc tccattacat gtgccttctg ccttatctag
     1261 tagcccgacg tatccagact tgcccttcat taggatctcc ccacaccgta atcccactgc
     1321 agcctcagag tcccccttca gccccccaca cccctacatc aacccatata tggactacat
     1381 ccgctcattg cacagcagcc catccctctc catgatctct gctgcccgag ggctgagccc
     1441 tacagatgct ccccatgctg gagtcagccc tgcggaatac tatcaccaga tggctctgct
     1501 gacaggccag cgcagccctt atgcagacat cattccctca gctgccactg ctggtgcagg
     1561 ggccatccat atggagtacc ttcatgccat ggacagcacc agatttccca gccctaggct
     1621 gtcagctagg cccagccgaa agcgcacact gtcaatatcg ccactgtcag atcatagctt
     1681 cgaccttcag accatgataa gaacatcccc taactccttg gttacaatcc tcaataattc
     1741 ccgtagcagc tcttcagcaa gtggttccta tgggcactta tcggcaagtg caatcagccc
     1801 tgcattgagc ttcacctacc cctccgctcc tgtgtctctt cacatgcatc aacagatcct
     1861 aagccgacag caaagcttag gctccgcatt cggacacagc cctcctctca tccaccctgc
     1921 tccaacattt ccaacacaga gacctatccc tgggattccg acagttctga accccgtcca
     1981 ggtcagctat ggcccttctg agtcctcaca gagcaagccc acaagcgagt ctgcagtgag
     2041 cagcactggt gaccctatgc ataataagcg gtccaagatc aagcctgatg aagacctccc
     2101 cagcccaggg tcacggggcc agcaggaaca gccggaagga acaaccctag tcaaggagga
     2161 agcggacaaa gatgaaagca agcaggagcc tgaagtcatc tacgagacaa actgccactg
     2221 ggaaggctgc accagagagt tcgacaccca agatcagctt gtgcatcata tcaataatga
     2281 ccacattcat ggagaaaaga aggaattcgt gtgccttgcc gagggtaccc gaatcttcga
     2341 tccggtcact ggtacaacgc atcgcatcga ggatgttgtc gatgggcgca agcctattca
     2401 tgtcgtggct gctgccaagg acggaacgct gctcgcgcgg cccgtggtgt cctggttcga
     2461 ccagggaacg cgggatgtga tcgggttgcg gatcgccggt ggcgccatcg tgtgggcgac
     2521 acccgatcac aaggtgctga cagagtacgg ctggcgtgcc gccggggaac tccgcaaggg
     2581 agacagggtg gccggaccgg gtggttctgg taacagcctg gccttgtccc tgacggccga
     2641 ccagatggtc agtgccttgt tggatgctga gccccccata ctctattccg agtatgatcc
     2701 taccagtccc ttcagtgaag cttcgatgat gggcttactg accaacctgg cagacaggga
     2761 gctggttcac atgatcaact gggcgaagag ggtgccaggc tttgtggatt tgaccctcca
     2821 tgatcaggcc caccttctag aacgtgcctg gctagagatc ctgatgattg gtctcgtctg
     2881 gcgctccatg gagcacccag ggaagctact gtttgctcct aacttgctct tggacaggaa
     2941 ccagggaaaa tgtgtagagg gcatggtgga gatcttcgac atgctgctgg ctacatcatc
     3001 tcggttccgc atgatgaatc tgcagggaga ggagtttgtg tgcctcaaat ctattatttt
     3061 gcttaattct ggagtgtaca catttctgtc cagcaccctg aagtctctgg aagagaagga
     3121 ccatatccac cgagccctgg acaagatcac ggacactttg atccacctga tggccaaggc
     3181 aggcctgacc ctgcagcagc agcaccagcg gctggcccag ctcctcctca tcctctccca
     3241 catcaggcac atgagtaaca aaggaatgga gcatctgtac agcatgaagt acaagaacgt
     3301 ggtgcccctc tatgacctgc tgctggagat gctggacgcc caccgcctac atgcgggtgg
     3361 ttctggtgct agccgcgtgc aggcgttcgc ggatgccctg gatgacaaat tcctgcacga
     3421 catgctggcg gaaggactcc gctattccgt gatccgagaa gtgctgccaa cgcggcgggc
     3481 acgaacgttc gacctcgagg tcgaggaact gcacaccctc gtcgccgaag gggttgtcgt
     3541 gcacaactgc cgctggcttg attgttctcg agagcagaaa ccgttcaaag cccagtacat
     3601 gttggtagtg catatgagaa gacacactgg ggagaagcct cacaaatgta catttgaagg
     3661 ttgcacaaaa gcctactcaa gactcgaaaa cctgaaaacc cacttgagat ctcacactgg
     3721 agagaagcca tacgtctgtg agcatgaggg ctgcaacaag gctttctcta atgcttcaga
     3781 tcgggccaag caccaaaaca gaacacattc caatgagaaa ccgtatgtat gcaaaatccc
     3841 tggctgcacc aagcgttaca cagaccccag ctctctccgg aaacacgtga agactgtgca
     3901 tggccctgag gctcatgtta ccaagaagca gcgtggggac atgcaccctc ggcctccacc
     3961 cccgagagat tccggcagcc attcacagtc caggtcccct ggccggccaa ctcagggagc
     4021 atttggtgag cagaaggagc tgagcaacac tacctcaaag cgggaagagt gcctccaggt
     4081 gaagactgtc aaggctgaga agcccatgac atctcagcca agccctggtg gtcagtcttc
     4141 atgcagcagc caacagtccc ccatcagcaa ctattccaac agtgggctcg agcttcctct
     4201 gactgatgga ggtagtgtag cagactctag aggatcccgg gctgactaca aagaccatga
     4261 cggtgattat aaagatcatg acatcgacta caaggatgac gatgacaagt agtgatcccg
     4321 ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag
     4381 tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
     4441 tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
     4501 cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc
     4561 tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
     4621 tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
     4681 gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
     4741 ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
     4801 ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg
     4861 cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca
     4921 ctcagtagat gcctgttgaa ttgggtacgc ggccagcttg gctgtggaat gtgtgtcagt
     4981 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5041 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5101 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     5161 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5221 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5281 gaggaattga tcagcttggg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     5341 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     5401 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5461 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     5521 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5581 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5641 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5701 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5761 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5821 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5881 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5941 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     6001 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     6061 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     6121 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     6181 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     6241 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     6301 ccgggctcga tcccctcgcg agttggttca gctgctgcct gaggctggac gacctcgcgg
     6361 agttctaccg gcagtgcaaa tccgtcggca tccaggaaac cagcagcggc tatccgcgca
     6421 tccatgcccc cgaactgcag gagtggggag gcacgatggc cgctttggtc gacccggacg
     6481 ggacgctcct gcgcctgata cagaacgaat tgcttgcagg catctcatga gtgtgtcttc
     6541 ccgttttccg cctgaggtca ctgcgtggat ggagcgctgg cgcctgctgc gcgacggcga
     6601 gctgctcacc acccactcgc caagctggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6661 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6721 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6781 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6841 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6901 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6961 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7021 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7081 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7141 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7201 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7261 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7321 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7381 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7441 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7501 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7561 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7621 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7681 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7741 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7801 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7861 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7921 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7981 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8041 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8101 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8161 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8221 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8281 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8341 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8401 gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     8461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     8521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     8581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     8641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     8701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     8761 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     8821 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg
     8881 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     8941 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     9001 cgttgtaaaa cgacggccag tgccaagctg atcta
//
