LOCUS       sequence-446225-        5459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hisx6-Tagged mStayGold(J) in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5459)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5459)
  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            "Unpublished"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-mStayGold(J)
FEATURES             Location/Qualifiers
     source          1..5459
                     /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             166..843
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     terminator      916..1010
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1054..1713
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1120..1139)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1778..1864
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1957..3039)
                     /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     3001..3020
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3040..3117)
                     /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(3201..3223)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3309..3449
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3364..3383
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3464..3481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3635..4223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3715..3734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4394..5185,5186..5254))
                     /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     5017..5036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5255..5359)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5427..5445
                     /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 gtctacaggc
      181 gaggagctgt ttaccggcgt ggtgcccttc aagttccagc tgaagggcac catcaacggc
      241 aagagcttca ccgtggaagg cgagggcgag ggcaatagcc acgagggcag ccacaaaggc
      301 aagtacgtgt gcaccagcgg caaactgcca atgtcttggg ccgccctggg aactagcttc
      361 ggctatggca tgaagtacta caccaagtac cccagcggcc tgaagaactg gttccacgag
      421 gtgatgcccg agggcttcac ctacgacaga cacatccagt acaagggcga cggcagcatc
      481 cacgccaagc accagcactt catgaagaac ggcacctacc acaacatcgt ggagttcacc
      541 ggccaggact tcaaggagaa cagccccgtg ctgaccggcg acatggacgt gagcctgccc
      601 aacgaggtgc agcacatccc catagatgac ggcgtggagt gcacagtgac cctgcagtac
      661 cctctgctga gcgacgagag caagtgcgtg gaggcctacc agaacaccat catcaagccc
      721 ctgcacaatc agccagcccc cgatgtgcca ttccactgga tcagaaagca gtacacccag
      781 agcaaggacg acaccgagga gagagaccac atcatccaga gcgagaccct ggaggcccac
      841 ctgtaaagcg gccgcgactc tagatcactc ggtaccccgg gtcgacctgc agccaagctt
      901 aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
      961 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc
     1021 ttggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     1081 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     1141 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     1201 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1261 catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1321 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1381 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1441 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1501 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1561 gttttcacta tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1621 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1681 cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct
     1741 taaacgcctg gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc
     1801 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     1861 aaatccgccc tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc
     1921 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1981 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2041 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     2101 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     2161 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     2221 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     2281 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     2341 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     2401 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     2461 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     2521 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     2581 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     2641 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     2701 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     2761 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     2821 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     2881 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     2941 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     3001 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     3061 ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcgga
     3121 atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt
     3181 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3241 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3301 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     3361 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     3421 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5401 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
