LOCUS       sequence-231230-        5558 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 5558)
  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 5558)
  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_LSSmOrange
FEATURES             Location/Qualifiers
     source          1..5558
                     /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             207..917
                     /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     620..639
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(919..943)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      965..1059
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1153..1812
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1219..1238)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1877..1963
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2056..3138)
                     /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     3100..3119
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3139..3216)
                     /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(3300..3322)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3408..3548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3463..3482
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3563..3580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3734..4322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3814..3833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4493..5284,5285..5353))
                     /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     5116..5135
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5354..5458)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5526..5544
                     /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 cgaggagaat aacatggcca
      241 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
      301 tcgagatcga gggcgagggc gagggccgcc cctacgaggg ctttcagacc gttaagctga
      361 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cttgtcccct cagttcacct
      421 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacctc aagctgtcct
      481 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
      541 tgactcagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
      601 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggcatg gaggcctcct
      661 ccgagcggat gtaccccgag gacggcgccc tgaagggcga ggacaagctc aggctgaagc
      721 tgaaggacgg cggccactac acctccgagg tcaagaccac ctacaaggcc aagaagcccg
      781 tgcagttgcc cggcgcctac atcgtcgaca tcaagttgga catcacctcc cacaacgagg
      841 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
      901 acgagctgta caagtaagac ccagctttct tgtacaaagt ggtaagctta attagctgag
      961 cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga
     1021 acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagtaacaa caccatttaa
     1081 atggagtggt tacaaatgga gtggttaatt aaggctagct tggcgagatt ttcaggagct
     1141 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     1201 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     1261 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     1321 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca
     1381 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     1441 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     1501 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     1561 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     1621 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     1681 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     1741 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     1801 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact
     1861 ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1921 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagattacg
     1981 tgcagtcgat gataagctgt caaacatgag aattgtgcct aatgagtgag ctaacttaca
     2041 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2101 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     2161 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     2221 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     2281 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     2341 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     2401 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     2461 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     2521 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     2581 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     2641 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     2701 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     2761 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     2821 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     2881 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     2941 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     3001 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     3061 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     3121 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     3181 cataccgcga aaggttttgc accattcgat ggtgtcggaa tttcgggcag cgttgggtcc
     3241 tggccacggg tgcgcatgat ctagagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     3301 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3361 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     3421 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     3481 tactgagagt gcaccacatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3541 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3601 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3661 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3901 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4141 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4381 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4441 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4501 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4561 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4621 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4681 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4741 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4801 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4861 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4921 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4981 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5041 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5101 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5161 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5221 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5281 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5341 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5401 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5461 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5521 ataaaaatag gcgtatcacg aggccctttc gtcttcac
//
