LOCUS       sequence-231176-        6308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fluorescent protein expression with an N-terminal His-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6308)
  AUTHORS   Holzapfel HY, Stern AD, Bouhaddou M, Anglin CM, Putur D, Comer S, 
            Birtwistle MR
  TITLE     Fluorescence Multiplexing with Spectral Imaging and Combinatorics.
  JOURNAL   ACS Comb Sci. 2018 Nov 12;20(11):653-659. doi: 
            10.1021/acscombsci.8b00101. Epub 2018 Oct 30.
  PUBMED    30339749
REFERENCE   2  (bases 1 to 6308)
  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"; doi: 
            "10.1021/acscombsci.8b00101"; journalName: "ACS Comb Sci"; date: 
            "2018-11-12- 12"; volume: "20"; issue: "11"; pages: "653-659"
COMMENT     Sequence Label: pQLinkHD_EBFP2-LSSmOrange
FEATURES             Location/Qualifiers
     source          1..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(36..50,51..56,57..73,74..80)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    56..72
                     /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    88..104
                     /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             123..134
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             147..167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    180..204
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(207..209,210..212,213..923)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(252..273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             951..1661
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1364..1383
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1669..1693)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      1715..1809
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1903..2562
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1969..1988)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2627..2713
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2806..3888)
                     /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     3850..3869
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3889..3966)
                     /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(4050..4072)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4158..4298
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4213..4232
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4313..4330)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4484..5072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4564..4583)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5243..6034,6035..6103))
                     /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     5866..5885
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6104..6208)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6276..6294
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagctta attaacaaca ccatttgtcg agaaatcata aaaaatttat ttgctttgtg
       61 agcggataac aattataata gattcaattg tgagcggata acaatttcac acagaattca
      121 ttaaagagga gaaattaact atgaaacatc accatcacca tcaccatggt gctgcaggta
      181 caagtttgta caaaaaagca ggcttcatgg tgagcaaggg cgaggagctg ttcaccgggg
      241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgaggg
      301 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
      361 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gagccacggc gtgcagtgct
      421 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      481 gctacgtcca ggagcgcacc atcttcttca aggacgacgg cacctacaag acccgcgccg
      541 aggtgaagtt cgagggcgac accctagtga accgcatcga gctgaagggc gtcgacttca
      601 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacatct
      661 atatcatggc cgtcaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaacg
      721 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
      781 gccccgtgct gctgcccgac agccactacc tgagcaccca gtccgtgctg agcaaagacc
      841 ccaacgagaa gcgcgatcac atggtcctgc tggagttccg caccgccgcc gggatcactc
      901 tcggcatgga cgagctgtac aagaccggtg ccggaggtgg gggcctagga atggtgagca
      961 agggcgagga gaataacatg gccatcatca aggagttcat gcgcttcaag gtgcgcatgg
     1021 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1081 agggctttca gaccgttaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1141 acatcttgtc ccctcagttc acctacggct ccaaggccta cgtgaagcac cccgccgaca
     1201 tccccgacta cctcaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1261 tcgaggacgg cggcgtggtg accgtgactc aggactcctc cctgcaggac ggcgagttca
     1321 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1381 agaccatggg catggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     1441 gcgaggacaa gctcaggctg aagctgaagg acggcggcca ctacacctcc gaggtcaaga
     1501 ccacctacaa ggccaagaag cccgtgcagt tgcccggcgc ctacatcgtc gacatcaagt
     1561 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1621 gccgccactc caccggaggc atggacgagc tgtacaagta aaagcttgac ccagctttct
     1681 tgtacaaagt ggtaagctta attagctgag cttggactcc tgttgataga tccagtaatg
     1741 acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt
     1801 ggtgagaatc caagtaacaa caccatttaa atggagtggt tacaaatgga gtggttaatt
     1861 aaggctagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     1921 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     1981 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     2041 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     2101 atgaatgctc atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat
     2161 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     2221 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     2281 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     2341 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     2401 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     2461 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     2521 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat
     2581 tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa
     2641 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     2701 gagtaggaca aatccgccct ctagattacg tgcagtcgat gataagctgt caaacatgag
     2761 aattgtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     2821 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2881 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     2941 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3001 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     3061 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     3121 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     3181 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     3241 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     3301 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     3361 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     3421 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     3481 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     3541 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     3601 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     3661 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     3721 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     3781 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     3841 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     3901 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat
     3961 ggtgtcggaa tttcgggcag cgttgggtcc tggccacggg tgcgcatgat ctagagctgc
     4021 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     4081 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     4141 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     4201 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccacatg cggtgtgaaa
     4261 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     4321 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4381 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4441 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4501 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4561 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4621 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4681 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4741 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4801 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4861 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4921 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4981 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5041 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5101 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5161 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5221 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5281 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5341 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5401 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5461 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5521 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5581 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5641 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5701 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5761 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5821 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5881 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5941 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6001 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6061 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6121 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6181 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     6241 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6301 gtcttcac
//
