LOCUS       sequence-445670-        5498 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hisx6-Tagged EYFP in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5498)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5498)
  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            "Unpublished"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: pQE81XN-EYFP
FEATURES             Location/Qualifiers
     source          1..5498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      160..169
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(166..168,169..171,172..885)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(211..232)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(472..491)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     819..840
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      955..1049
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1093..1752
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1159..1178)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1817..1903
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1996..3078)
                     /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     3040..3059
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3079..3156)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3240..3262)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3348..3488
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3403..3422
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3503..3520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3674..4262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3754..3773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4433..5224,5225..5293))
                     /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     5056..5075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5294..5398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5466..5484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatctcacc atcaccatca ccatacggat ccactagtcg ccaccatggt gagcaagggc
      181 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      241 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      301 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
      361 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
      421 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
      481 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
      541 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
      601 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
      661 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
      721 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
      781 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
      841 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct
      901 agatcactcg gtaccccggg tcgacctgca gccaagctta attagctgag cttggactcc
      961 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
     1021 gccgccgggc gttttttatt ggtgagaatc caagctagct tggcgagatt ttcaggagct
     1081 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     1141 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     1201 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     1261 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca
     1321 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     1381 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     1441 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     1501 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     1561 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcactat gggcaaatat
     1621 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     1681 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     1741 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact
     1801 ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1861 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagattacg
     1921 tgcagtcgat gataagctgt caaacatgag aattgtgcct aatgagtgag ctaacttaca
     1981 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2041 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     2101 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     2161 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     2221 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     2281 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     2341 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     2401 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     2461 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     2521 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     2581 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     2641 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     2701 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     2761 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     2821 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     2881 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     2941 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     3001 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     3061 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     3121 cataccgcga aaggttttgc accattcgat ggtgtcggaa tttcgggcag cgttgggtcc
     3181 tggccacggg tgcgcatgat ctagagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     3241 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3301 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     3361 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     3421 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3481 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3541 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3601 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3661 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3721 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3781 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3841 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3901 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3961 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4021 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4081 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4141 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4201 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4261 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4321 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4381 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4441 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4501 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4561 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4621 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4681 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4741 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4801 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4861 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4921 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4981 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5041 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5101 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5161 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5221 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5281 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5341 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5401 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5461 ataaaaatag gcgtatcacg aggccctttc gtcttcac
//
