LOCUS       sequence-231174-        5567 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 5567)
  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 5567)
  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
FEATURES             Location/Qualifiers
     source          1..5567
                     /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..926)
                     /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(928..952)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      974..1068
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1162..1821
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1228..1247)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1886..1972
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2065..3147)
                     /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     3109..3128
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3148..3225)
                     /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(3309..3331)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3417..3557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3472..3491
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3572..3589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3743..4331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3823..3842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4502..5293,5294..5362))
                     /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     5125..5144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5363..5467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5535..5553
                     /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 aagtaagacc cagctttctt gtacaaagtg gtaagcttaa
      961 ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat
     1021 ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aagtaacaac
     1081 accatttaaa tggagtggtt acaaatggag tggttaatta aggctagctt ggcgagattt
     1141 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
     1201 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     1261 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     1321 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattt
     1381 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
     1441 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
     1501 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     1561 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
     1621 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     1681 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     1741 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
     1801 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggg
     1861 gtaatgactc tctagcttga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1921 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccctc
     1981 tagattacgt gcagtcgatg ataagctgtc aaacatgaga attgtgccta atgagtgagc
     2041 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2101 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     2161 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     2221 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     2281 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     2341 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     2401 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     2461 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     2521 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     2581 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     2641 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     2701 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     2761 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     2821 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     2881 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     2941 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     3001 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     3061 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     3121 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     3181 ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcggaat ttcgggcagc
     3241 gttgggtcct ggccacgggt gcgcatgatc tagagctgcc tcgcgcgttt cggtgatgac
     3301 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     3361 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     3421 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     3481 agcagattgt actgagagtg caccacatgc ggtgtgaaat accgcacaga tgcgtaagga
     3541 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3601 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3661 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3721 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3781 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3841 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3901 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3961 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4021 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4081 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4141 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4201 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4261 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4321 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4381 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4441 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4501 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4561 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4621 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4681 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4741 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4801 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4861 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4921 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4981 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5041 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5101 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5161 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5221 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5281 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5341 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5401 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5461 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5521 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcac
//
