LOCUS       sequence-413580-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human NKX6-2 T252D phosphoresistant mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  AUTHORS   Ferreira-Peralta P, Franca B, Murtinheira F, Rodrigues MS, Herrera F
  TITLE     Is spastic ataxia 8 a protein misfolding disorder?
  JOURNAL   Biochim Biophys Acta Mol Basis Dis. 2023 Sep 9;1870(1):166882. doi: 
            10.1016/j.bbadis.2023.166882.
  PUBMED    37696463
REFERENCE   2  (bases 1 to 6943)
  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"; journalName: "Biochim 
            Biophys Acta Mol Basis Dis."; date: "2023-09-9"; volume: 
            "1870(1):166882. doi"; pages: " 10.1016/j.bbadis.2023.166882"
COMMENT     Sequence Label: pcDNA3.1 NKX6-2-Venus S246A/T252A
FEATURES             Location/Qualifiers
     source          1..6943
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2086..2105)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2534..2551)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2540..2764
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2810..3238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2897..2916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3107..3128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3247..3267)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3252..3581
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3432..3567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3494..3513
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3648..4442
                     /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(3702..3721)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4312..4331
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4616..4737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4653..4672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4707..4726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4786..4802)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4786..4802)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4799..4821)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4810..4826
                     /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(4834..4840,4841..4858,4859..4864))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4879..4900
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5017..5034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5188..5776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5268..5287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5947..6738,6739..6807))
                     /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     6570..6589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6808..6912)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gacaccatgg acacaaacag acctggcgcc tttgtgctgt ctagcgctcc tctggccgct
      961 ctgcacaata tggccgagat gaagaccagc ctgtttccat acgctctgca gggacctgcc
     1021 ggctttaagg ctcctgctct tggaggactg ggagcacaac tgcctctggg aacacctcac
     1081 ggcatcagcg atattctggg cagacctgtt ggagctgctg gcggaggatt gctcggagga
     1141 ctgcctagac tgaatggact ggcttcttct gccggcgtgt actttggacc tgctgctgcc
     1201 gttgccagag gctatcctaa acctctggct gagcttcccg gcagacctcc tattttttgg
     1261 cctggtgttg tgcagggcgc tccttggaga gatcctagac ttgctggacc agctcctgca
     1321 ggcggagtgc tggataagga tggcaagaag aagcacagca gacccacctt cagcggccag
     1381 cagatctttg ccctggaaaa gaccttcgag cagaccaagt atctggctgg ccctgagaga
     1441 gccagactgg cctattctct gggcatgacc gagagccaag tgaaagtgtg gttccagaac
     1501 agacggacca agtggcggaa gagacatgcc gtggaaatgg ccagcgccaa gaagaaacag
     1561 gacagcgacg ccgagaagct gaaagttgga ggctccgatg ccgaggacga cgacgagtat
     1621 aatagacctc tggaccccaa cgccgacgat gagaagatcg cccggctgct gaagaaacac
     1681 aagcccagca atctggccct ggtgtctcct tgtggtggcg gagctgggga tgcacttgga
     1741 tccggcggag gtggaagcgg cggaggcgga tctggtacca tggtttctaa gggcgaagaa
     1801 ctgttcaccg gcgtggtgcc tatcctggtg gaactggatg gggatgtgaa cggccacaag
     1861 ttctctgtgt ctggcgaagg cgaaggggat gccacatacg gcaagctgac cctgaagctg
     1921 atctgcacca caggcaaact gcccgtgcct tggcctacac tggtcacaac actcggctac
     1981 ggcctgcagt gctttgccag atatcccgac cacatgaagc agcacgattt cttcaagagc
     2041 gccatgcctg agggctacgt gcaagagcgg accatcttct tcaaggacga cggcaactac
     2101 aagaccaggg ccgaagtgaa gttcgagggc gacaccctgg tcaacagaat cgagctgaag
     2161 ggcatcgact ttaaagagga cggcaacatc ctgggccaca agctcgagta caactacaac
     2221 agccacaacg tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     2281 atccggcaca acatcgagga tggcggcgtg cagctggccg atcactatca gcagaacacc
     2341 cctatcggag atggccccgt cctgctgcct gataaccact acctgagcta ccagtctgcc
     2401 ctgagcaagg accccaatga gaagagggat cacatggtgc tgctggaatt cgtgacagcc
     2461 gccggaatca cactcggcat ggacgagctg tacaaatgag gatcctaagg gcccgtttaa
     2521 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2581 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2641 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2701 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2761 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2821 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2881 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2941 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3001 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3061 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3121 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3181 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3241 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3301 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3361 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3421 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3481 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3541 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3601 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3661 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3721 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3781 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3841 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3901 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3961 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4021 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4081 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4141 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4201 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4261 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4321 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4381 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4441 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4501 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4561 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4621 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4681 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4741 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4801 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4861 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4921 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4981 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5041 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5101 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5161 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5221 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5281 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5341 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5401 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5461 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5521 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5581 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5641 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5701 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5761 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5821 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5881 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5941 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6001 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6061 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6121 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6181 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6241 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6301 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6361 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6421 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6481 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6541 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6601 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6661 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6721 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6781 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6841 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6901 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
