LOCUS       sequence-291736-        6533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For making TNKS-2-M1054V RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6533)
  AUTHORS   Williams C
  TITLE     Williams Lab Plasmids 2020
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6533)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGEMHE-TNKS-2-M1054V
FEATURES             Location/Qualifiers
     source          1..6533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           51..93
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      126..135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             135..158
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1731..1742
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           3704..3829
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(3875..3893)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3876..3893)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3911..3927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3911..3927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3924..3946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3935..3951
                     /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(3959..3965,3966..3983,3984..3989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4004..4025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4142..4159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4313..4901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4393..4412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5072..5863,5864..5932))
                     /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     5695..5714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5933..6037)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6105..6123
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6161..6183)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6283..6302
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6496..6518
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6510..6527
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6511..6527
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattaatt cgagctcggt acccagcttg cttgttcttt
       61 ttgcagaagc tcagaataaa cgctcaactt tggcagatca attccccggt gctagcgcta
      121 ccggtgccac catggactac aaagacgatg acgacaagct tgcggccgcg tcgggtcgcc
      181 gctgcgccgg cgggggagcg gcctgcgcga gcgccgcggc cgaggccgtg gagccggccg
      241 cccgagagct gttcgaggcg tgccgcaacg gggacgtgga acgagtcaag aggctggtga
      301 cgcctgagaa ggtgaacagc cgcgacacgg cgggcaggaa atccaccccg ctgcacttcg
      361 ccgcaggttt tgggcggaaa gacgtagttg aatatttgct tcagaatggt gcaaatgtcc
      421 aagcacgtga tgatgggggc cttattcctc ttcataatgc atgctctttt ggtcatgctg
      481 aagtagtcaa tctccttttg cgacatggtg cagaccccaa tgctcgagat aattggaatt
      541 atactcctct ccatgaagct gcaattaaag gaaagattga tgtttgcatt gtgctgttac
      601 agcatggagc tgagccaacc atccgaaata cagatggaag gacagcattg gatttagcag
      661 atccatctgc caaagcagtg cttactggtg aatataagaa agatgaactc ttagaaagtg
      721 ccaggagtgg caatgaagaa aaaatgatgg ctctactcac accattaaat gtcaactgcc
      781 acgcaagtga tggcagaaag tcaactccat tacatttggc agcaggatat aacagagtaa
      841 agattgtaca gctgttactg caacatggag ctgatgtcca tgctaaagat aaaggtgatc
      901 tggtaccatt acacaatgcc tgttcttatg gtcattatga agtaactgaa cttttggtca
      961 agcatggtgc ctgtgtaaat gcaatggact tgtggcaatt cactcctctt catgaggcag
     1021 cttctaagaa cagggttgaa gtatgttctc ttctcttaag ttatggtgca gacccaacac
     1081 tgctcaattg tcacaataaa agtgctatag acttggctcc cacaccacag ttaaaagaaa
     1141 gattagcata tgaatttaaa ggccactcgt tgctgcaagc tgcacgagaa gctgatgtta
     1201 ctcgaatcaa aaaacatctc tctctggaaa tggtgaattt caagcatcct caaacacatg
     1261 aaacagcatt gcattgtgct gctgcatctc catatcccaa aagaaagcaa atatgtgaac
     1321 tgttgctaag aaaaggagca aacatcaatg aaaagactaa agaattcttg actcctctgc
     1381 acgtggcatc tgagaaagct cataatgatg ttgttgaagt agtggtgaaa catgaagcaa
     1441 aggttaatgc tctggataat cttggtcaga cttctctaca cagagctgca tattgtggtc
     1501 atctacaaac ctgccgccta ctcctgagct atgggtgtga tcctaacatt atatcccttc
     1561 agggctttac tgctttacag atgggaaatg aaaatgtaca gcaactcctc caagagggta
     1621 tctcattagg taattcagag gcagacagac aattgctgga agctgcaaag gctggagatg
     1681 tcgaaactgt aaaaaaactg tgtactgttc agagtgtcaa ctgcagagac attgaagggc
     1741 gtcagtctac accacttcat tttgcagctg ggtataacag agtgtccgtg gtggaatatc
     1801 tgctacagca tggagctgat gtgcatgcta aagataaagg aggccttgta cctttgcaca
     1861 atgcatgttc ttatggacat tatgaagttg cagaacttct tgttaaacat ggagcagtag
     1921 ttaatgtagc tgatttatgg aaatttacac ctttacatga agcagcagca aaaggaaaat
     1981 atgaaatttg caaacttctg ctccagcatg gtgcagaccc tacaaaaaaa aacagggatg
     2041 gaaatactcc tttggatctt gttaaagatg gagatacaga tattcaagat ctgcttaggg
     2101 gagatgcagc tttgctagat gctgccaaga agggttgttt agccagagtg aagaagttgt
     2161 cttctcctga taatgtaaat tgccgcgata cccaaggcag acattcaaca cctttacatt
     2221 tagcagctgg ttataataat ttagaagttg cagagtattt gttacaacac ggagctgatg
     2281 tgaatgccca agacaaagga ggacttattc ctttacataa tgcagcatct tacgggcatg
     2341 tagatgtagc agctctacta ataaagtata atgcatgtgt caatgccacg gacaaatggg
     2401 ctttcacacc tttgcacgaa gcagcccaaa agggacgaac acagctttgt gctttgttgc
     2461 tagcccatgg agctgacccg actcttaaaa atcaggaagg acaaacacct ttagatttag
     2521 tttcagcaga tgatgtcagc gctcttctga cagcagccat gcccccatct gctctgccct
     2581 cttgttacaa gcctcaagtg ctcaatggtg tgagaagccc aggagccact gcagatgctc
     2641 tctcttcagg tccatctagc ccatcaagcc tttctgcagc cagcagtctt gacaacttat
     2701 ctgggagttt ttcagaactg tcttcagtag ttagttcaag tggaacagag ggtgcttcca
     2761 gtttggagaa aaaggaggtt ccaggagtag attttagcat aactcaattc gtaaggaatc
     2821 ttggacttga gcacctaatg gatatatttg agagagaaca gatcactttg gatgtattag
     2881 ttgagatggg gcacaaggag ctgaaggaga ttggaatcaa tgcttatgga cataggcaca
     2941 aactaattaa aggagtcgag agacttatct ccggacaaca aggtcttaac ccatatttaa
     3001 ctttgaacac ctctggtagt ggaacaattc ttatagatct gtctcctgat gataaagagt
     3061 ttcagtctgt ggaggaagag atgcaaagta cagttcgaga gcacagagat ggaggtcatg
     3121 caggtggaat cttcaacaga tacaatattc tcaagattca gaaggtttgt aacaagaaac
     3181 tatgggaaag atacactcac cggagaaaag aagtttctga agaaaaccac aaccatgcca
     3241 atgaacgaat gctatttcat gggtctcctt ttgtgaatgc aattatccac aaaggctttg
     3301 atgaaaggca tgcgtacata ggtggtgtgt ttggagctgg catttatttt gctgaaaact
     3361 cttccaaaag caatcaatat gtatatggaa ttggaggagg tactgggtgt ccagttcaca
     3421 aagacagatc ttgttacatt tgccacaggc agctgctctt ttgccgggta accttgggaa
     3481 agtctttcct gcagttcagt gcaatgaaaa tggcacattc tcctccaggt catcactcag
     3541 tcactggtag gcccagtgta aatggcctag cattagctga atatgttatt tacagaggag
     3601 aacaggctta tcctgagtat ttaattactt accagattat gaggcctgaa ggtatggtcg
     3661 atggataatc tagataaatg atagcttgat ctggttacca ctaaaccagc ctcaagaaca
     3721 cccgaatgga gtctctaagc tacataatac caacttacac tttacaaaat gttgtccccc
     3781 aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag tttcttcaca kkcgcgccgc
     3841 cggcgattta aattaattaa tctagcttga gtattctata gtgtcaccta aatagcttgg
     3901 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3961 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4141 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4201 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4261 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4321 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4381 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4441 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4501 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4561 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4621 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4681 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4741 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4801 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4861 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4921 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4981 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5041 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5101 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5161 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5221 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5281 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5341 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5401 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5461 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5521 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5581 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5641 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5701 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5761 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5821 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5881 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5941 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6001 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6061 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     6121 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     6181 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     6241 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     6301 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     6361 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6421 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     6481 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttg
//
