LOCUS       sequence-473833-        6020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TGP-E, a thermostable fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6020)
  AUTHORS   Anderson MR, Dargatz CJ, Banerjee T, DeVore NM
  TITLE     Green, yellow, or cyan? Introduction of color change mutations into 
            a green thermostable fluorescent protein and characterization during
            an introduction to biochemistry lab course.
  JOURNAL   Biochem Mol Biol Educ. 2024 Sep-Oct;52(5):549-558. doi: 
            10.1002/bmb.21841. Epub 2024 Jun 8.
  PUBMED    38850239
REFERENCE   2  (bases 1 to 6020)
  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: "Biochem Mol
            Biol Educ. 2024 Sep-Oct"; volume: "52(5)"; pages: "549-558. doi: 
            10.1002/bmb.21841. Epub 2024 Jun 8"
COMMENT     Sequence Label: pETCK3 TGP-E
FEATURES             Location/Qualifiers
     source          1..6020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             332..373
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             374..391
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             411..428
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             438..455
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(508..526)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      522..569
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      606..1061
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(693..712)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     903..924
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1153..1968)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1153..1968)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1876..1895
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1960..1979
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2090..2678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2579..2598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2832..2849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2864..3003
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2929..2948)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3089..3111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3105..3296)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4105..5187)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5149..5168
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5188..5265)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5468..5487
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5571..5590
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5571..5589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5590..5614
                     /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)."
     RBS             5629..5651
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aattcgcttt gatggaacta acttcccccc gaacggccct gtaatgcaaa agaagacctt
       61 aaaatgggaa cctagcaccg aaaaaatgta tgtagaagac ggagttctta agggtgacgt
      121 agaaatggca cttctgctcg aaggaggtgg acactaccgc tgcgatttta aaaccactta
      181 taaagccaaa aaagatgttc gtcttccaga tgcacacgag gtggaccacc gcattgaaat
      241 cctgagccac gataaagatt ataataaagt tagactctat gaacacgccg aagcccgcta
      301 ttctggcgga ggcagcggag gcggtgctag cggcaaacca atcccaaacc cactgctggg
      361 cctggatagt actcaccatc accatcacca ttaggcggcc gcttggatcc caccaccacc
      421 accaccacta actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
      481 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      541 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
      601 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      661 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      721 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      781 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      841 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      901 tagtggactc ttgttccaaa ctggaacaac actcaacccc atctcggtct attcttttga
      961 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1021 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1081 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1141 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1201 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1261 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1321 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1381 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     1441 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     1501 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1561 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1621 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1681 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1741 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1801 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     1861 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     1921 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     1981 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2161 gtttgccgga tcaagaacta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2221 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2701 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2761 attctgtgga taaccgtatt accgcctttg agtgagccga taccgctcgc cgcagccgaa
     2821 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2881 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     2941 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3001 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3061 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3121 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3181 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctcaag aagcgttaat
     3241 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3301 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3361 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3421 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3481 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3541 atccggaata taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3601 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3661 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3721 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     3781 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     3841 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctcaagcga
     3901 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     3961 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4021 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4081 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4141 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4201 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4261 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4321 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4381 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcacgcattg cgcccagcgc
     4441 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4501 ggtttgttga aaaccggaca tggcactcca atcgccttcc cgttccgcta tcggctgaat
     4561 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4621 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4681 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4741 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4801 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4861 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4921 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4981 agaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5041 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5101 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5161 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5221 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5281 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5341 ccgccgccgc aaggaatggt gccaaggaga tggcgcccaa cagtcccccg gccacggggc
     5401 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5461 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5521 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     5581 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     5641 taagaaggag atatacatat gggcgcgcat gcctcagtaa ttaaaccgga aatgaaaatt
     5701 aaattgcgta tggaaggtgc cgttaacggc cataaatttg taattgaagg agaaggaata
     5761 ggcaaaccat acgaaggaac ccagaccctg gatttaaccg tagaagaagg cgcacctctc
     5821 cctttctcgt acgacatcct caccccagcc ttcgaatacg gcaatcgcgc tttcaccaaa
     5881 tacccagaag atattccaga ctattttaaa caagcattcc ccgaaggcta ttcttgggaa
     5941 cgctctatga cctatgaaga tcaaggaatt tgtatcgcta cctccgacat tactatggaa
     6001 ggagactgtt ttttttatga
//
