LOCUS       sequence-232462-        9035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human protein Gli containing a 4HT-inducible intein 
            evolved to operate at 30C.
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 30R3-1
FEATURES             Location/Qualifiers
     source          1..9035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4098..4163
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4174..4796
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4373..4392)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4820..4840)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4838..5135
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5189..5983
                     /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(5243..5262)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5853..5872
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6512..7100)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6592..6611)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7271..8062,8063..8131))
                     /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     7894..7913
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8132..8236)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8263..8718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8350..8369)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8560..8581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8844..8866
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8858..8875
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8859..8875
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agaattaagc ttgcggccgc accatggagg cccaggccca cagctctacg gcgactgaga
      661 ggaagaaagc tgaaaattcc attgggaaat gtcccacgag aacagatgtc agcgagaagg
      721 ccgtggcctc tagtaccact tccaatgagg atgaaagtcc tggacagatc tatcaccgag
      781 agagaagaaa cgcaatcact atgcagcctc agagtgtgca gggtctcaac aaaatcagtg
      841 aggagccctc gacgtctagt gatgagaggg cctcgctgat caagaaagag atccatggct
      901 ctctaccaca tctggcggag ccctctctcc cttaccgtgg gactgtgttt gccatggatc
      961 cccggaatgg ctacatggag cctcactacc accctcctca tcttttccct gccttccatc
     1021 ctcctgtacc aattgatgcc agacatcatg agggccgtta ccattatgat ccatctccta
     1081 ttcctccatt acatgtgcct tctgccttat ctagtagccc gacgtatcca gacttgccct
     1141 tcattaggat ctccccacac cgtaatccca ctgcagcctc agagtccccc ttcagccccc
     1201 cacaccccta catcaaccca tatatggact acatccgctc attgcacagc agcccatccc
     1261 tctccatgat ctctgctgcc cgagggctga gccctacaga tgctccccat gctggagtca
     1321 gccctgcgga atactatcac cagatggctc tgctgacagg ccagcgcagc ccttatgcag
     1381 acatcattcc ctcagctgcc actgctggtg caggggccat ccatatggag taccttcatg
     1441 ccatggacag caccagattt cccagcccta ggctgtcagc taggcccagc cgaaagcgca
     1501 cactgtcaat atcgccactg tcagatcata gcttcgacct tcagaccatg ataagaacat
     1561 cccctaactc cttggttaca atcctcaata attcccgtag cagctcttca gcaagtggtt
     1621 cctatgggca cttatcggca agtgcaatca gccctgcatt gagcttcacc tacccctccg
     1681 ctcctgtgtc tcttcacatg catcaacaga tcctaagccg acagcaaagc ttaggctccg
     1741 cattcggaca cagccctcct ctcatccacc ctgctccaac atttccaaca cagagaccta
     1801 tccctgggat tccgacagtt ctgaaccccg tccaggtcag ctatggccct tctgagtcct
     1861 cacagagcaa gcccacaagc gagtctgcag tgagcagcac tggtgaccct atgcataata
     1921 agcggtccaa gatcaagcct gatgaagacc tccccagccc agggtcacgg ggccagcagg
     1981 aacagccgga aggaacaacc ctagtcaagg aggaagcgga caaagatgaa agcaagcagg
     2041 agcctgaagt catctacgag acaaactgcc actgggaagg ctgcaccaga gagttcgaca
     2101 cccaagatca gcttgtgcat catatcaata atgaccacat tcatggagaa aagaaggaat
     2161 tcgtgtgcct tgccgagggt acccgaatct tcgatccggt cactggtaca acgcatcgca
     2221 tcgaggatgt tgtcgatggg cgcaagccta ttcatgtcgt ggctgctgcc aaggacggaa
     2281 cgctgctcgc gcggcccgtg gtgtcctggt tcgaccaggg aacgcgggat gtgatcgggt
     2341 tgcggatcgc cggtggcgcc accgtgtggg cgacacccga tcacaaggtg ctgacagagt
     2401 acggctggcg tgccgccggg gaactccgca agggagacag ggtggccgga ccgggtggtt
     2461 ctggtaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg
     2521 ctgagccccc cataccctat tccgagtatg atcctaccag tcccttcagt gaagcttcga
     2581 tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga
     2641 agagggtgcc aggctttgtg gatttgaccc tccatgatca ggcccacctt ctagaatgtg
     2701 cctggctaga gatcctgatg attggtctcg tctggcgctc catggagcac ccagggaagc
     2761 tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg
     2821 tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg
     2881 gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc
     2941 tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagcc ctggacaaga
     3001 tcacggacac tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc
     3061 agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggaa
     3121 tggagcatct gtacagcatg aagtacaaga acgtggtgcc cctctatgac ctgctgctgg
     3181 agatgctgga cgcccaccgc ctacatgcgg gtggttctgg tgctagccgc gtgcaggcgt
     3241 tcgcggatgc cctggatgac aaattcctgc acgacatgct ggcggaagga ctccgctatt
     3301 ccgtgatccg agaagtgctg ccaacgcagc gggcacgaac gttcgacctc gaggtcgagg
     3361 aactgcacac cctcgtcgcc gaaggggttg tcgtgcacaa ctgccgctgg cttgattgtt
     3421 ctcgagagca gaaaccgttc aaagcccagt acatgttggt agtgcatatg agaagacaca
     3481 ctggggagaa gcctcacaaa tgtacatttg aaggttgcac aaaagcctac tcaagactcg
     3541 aaaacctgaa aacccacttg agatctcaca ctggagagaa gccatacgtc tgtgagcatg
     3601 agggctgcaa caaggctttc tctaatgctt cagatcgggc caagcaccaa aacagaacac
     3661 attccaatga gaaaccgtat gtatgcaaaa tccctggctg caccaagcgt tacacagacc
     3721 ccagctctct ccggaaacac gtgaagactg tgcatggccc tgaggctcat gttaccaaga
     3781 agcagcgtgg ggacatgcac cctcggcctc cacccccgag agattccggc agccattcac
     3841 agtccaggtc ccctggccgg ccaactcagg gagcatttgg tgagcagaag gagctgagca
     3901 acactacctc aaagcgggaa gagtgcctcc aggtgaagac tgtcaaggct gagaagccca
     3961 tgacatctca gccaagccct ggtggtcagt cttcatgcag cagccaacag tcccccatca
     4021 gcaactattc caacagtggg ctcgagcttc ctctgactga tggaggtagt gtagcagact
     4081 ctagaggatc ccgggctgac tacaaagacc atgacggtga ttataaagat catgacatcg
     4141 actacaagga tgacgatgac aagtagtgat cccgggtggc atccctgtga cccctcccca
     4201 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     4261 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     4321 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     4381 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     4441 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     4501 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     4561 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     4621 gcgtgaacca ctgctccctt ccctgtcctt ctgattttaa aataactata ccagcaggag
     4681 gacgtccaga cacagcatag gctacctggc catgcccaac cggtgggaca tttgagttgc
     4741 ttgcttggca ctgtcctctc atgcgttggg tccactcagt agatgcctgt tgaattgggt
     4801 acgcggccag cttggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4861 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4921 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4981 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5041 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     5101 gagctattcc agaagtagtg aggaggcttt tttggaggaa ttgatcagct tgggatctga
     5161 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     5221 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     5281 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     5341 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     5401 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     5461 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     5521 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     5581 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5641 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5701 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5761 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5821 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5881 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5941 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     6001 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     6061 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     6121 cgcggggatc tcatgctgga gttcttcgcc caccccgggc tcgatcccct cgcgagttgg
     6181 ttcagctgct gcctgaggct ggacgacctc gcggagttct accggcagtg caaatccgtc
     6241 ggcatccagg aaaccagcag cggctatccg cgcatccatg cccccgaact gcaggagtgg
     6301 ggaggcacga tggccgcttt ggtcgacccg gacgggacgc tcctgcgcct gatacagaac
     6361 gaattgcttg caggcatctc atgagtgtgt cttcccgttt tccgcctgag gtcactgcgt
     6421 ggatggagcg ctggcgcctg ctgcgcgacg gcgagctgct caccacccac tcgccaagct
     6481 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6541 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6601 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6661 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6721 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6781 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6841 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6901 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6961 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7021 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7081 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7141 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7201 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7261 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7321 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7381 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7441 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7501 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7561 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7621 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7681 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7741 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7801 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7861 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7921 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7981 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8041 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8101 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8161 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8221 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg
     8281 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     8341 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     8401 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     8461 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     8521 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     8581 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     8641 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     8701 tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     8761 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     8821 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
     8881 gctgatctat acattgaatc aatattggca attagccata ttagtcattg gttatatagc
     8941 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat
     9001 ttatattggc tcatgtccaa tatgaccgcc atgtt
//
