LOCUS       sequence-266443-        6230 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 6230)
  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 6230)
  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-ECFP
FEATURES             Location/Qualifiers
     source          1..6230
                     /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"
     protein_bind    complement(1591..1615)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      1637..1731
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1825..2484
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1891..1910)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2549..2635
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2728..3810)
                     /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     3772..3791
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3811..3888)
                     /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(3972..3994)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4080..4220
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4135..4154
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4235..4252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4406..4994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4486..4505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5165..5956,5957..6025))
                     /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     5788..5807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6026..6130)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6198..6216
                     /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 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1021 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca
     1081 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1141 acgtccagga gcgcaccatc ttcttcaagg gcgacggcaa ctacaagacc cgcgccgagg
     1201 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1261 aggacggcaa catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata
     1321 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
     1381 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1441 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1501 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1561 gcatggacga gctgtacaag taaaagcttg acccagcttt cttgtacaaa gtggtaagct
     1621 taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     1681 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagtaac
     1741 aacaccattt aaatggagtg gttacaaatg gagtggttaa ttaaggctag cttggcgaga
     1801 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     1861 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     1921 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     1981 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     2041 tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     2101 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     2161 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     2221 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     2281 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     2341 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     2401 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     2461 gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct
     2521 ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt cgaaagactg
     2581 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     2641 ctctagatta cgtgcagtcg atgataagct gtcaaacatg agaattgtgc ctaatgagtg
     2701 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2761 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2821 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     2881 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     2941 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     3001 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3061 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3121 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3181 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3241 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     3301 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     3361 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     3421 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     3481 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     3541 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     3601 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     3661 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     3721 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     3781 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     3841 gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg aatttcgggc
     3901 agcgttgggt cctggccacg ggtgcgcatg atctagagct gcctcgcgcg tttcggtgat
     3961 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     4021 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4081 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     4141 cagagcagat tgtactgaga gtgcaccaca tgcggtgtga aataccgcac agatgcgtaa
     4201 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5281 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6181 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
//
