LOCUS       sequence-355922-        6061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV overexpression of SNAP-tag fast fusion to H2B for nuclear 
            localization in mammalian cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6061)
  AUTHORS   Frei MS, Koch B, Hiblot J, Johnsson K
  TITLE     Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic 
            Fluorescent Probes.
  JOURNAL   ACS Chem Biol. 2022 Jun 17;17(6):1321-1327. doi: 
            10.1021/acschembio.2c00041. Epub 2022 May 18.
  PUBMED    35584304
REFERENCE   2  (bases 1 to 6061)
  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/acschembio.2c00041"; journalName: "ACS Chem Biol"; date: 
            "2022-06-17- 17"; volume: "17"; issue: "6"; pages: "1321-1327"
COMMENT     Sequence Label: pcDNA5/FRT/TO_H2B-STf
FEATURES             Location/Qualifiers
     source          1..6061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1043..1420
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(2013..2030)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2019..2243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2376..2395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2527..2574
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2582..3604
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2582..3604
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3734..3855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3771..3790)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3825..3844
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3904..3920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3904..3920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3917..3939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3928..3944
                     /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)."
     promoter        complement(join(3952..3958,3959..3976,3977..3982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3997..4018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4135..4152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4306..4894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4386..4405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5065..5856,5857..5925))
                     /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     5688..5707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5926..6030)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcaa ccatgccaga gccagcgaag tctgctcccg ccccgaaaaa
     1081 gggctccaag aaggcggtga ctaaggcgca gaagaaaggc ggcaagaagc gcaagcgcag
     1141 ccgcaaggag agctattcca tctatgtgta caaggttctg aagcaggtcc accctgacac
     1201 cggcatttcg tccaaggcca tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg
     1261 catcgcaggt gaggcttccc gcctggcgca ttacaacaag cgctcgacca tcacctccag
     1321 ggagatccag acggccgtgc gcctgctgct gcctggggag ttggccaagc acgccgtgtc
     1381 cgagggtact aaggccatca ccaagtacac cagcgctaag ggtggcgata aagactgcga
     1441 aatgaaacgc actaccctgg acagcccgct gggtaaactg gaactgagcg gttgcgagca
     1501 gggcctgcac cgcatcattt tcctgggcaa aggtacgagc gctgcggatg cggtcgaagt
     1561 accggctccg gctgcggttc tgggtggccc ggagccgctg atgcaggcta ccgcatggct
     1621 gaacgcatat tttcaccagc cagaggcgat tgaagagttc cctgtaccgg ccctgcatca
     1681 cccggtattt cagcaggaat cctttacccg tcaggtactg tggaaactgc tgaaagtagt
     1741 gaaattcggc gaggtgatct cctattccca tctggcggcg ctggctggca acccagcggc
     1801 aactgcggcg gtgaaaaccg ctctgtctgg taatccggtg ccaattctga tcccgtgcca
     1861 ccgtgtggtt cagggcgacc tggacgtagg tggctacgaa ggcggcctgg ctgtgaaaga
     1921 atggctgctg gcgcacgaag gtcaccgtct gggtaaacca ggcctgggct aaccgctcga
     1981 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2041 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2101 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2161 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2221 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2281 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2341 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2401 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2461 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2521 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
     2581 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     2641 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     2701 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     2761 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     2821 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     2881 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     2941 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     3001 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     3061 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     3121 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     3181 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     3241 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     3301 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     3361 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     3421 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     3481 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     3541 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     3601 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3661 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3721 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3781 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3841 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3901 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3961 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4021 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4081 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4141 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4201 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4261 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4321 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4381 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4441 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4501 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4561 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4621 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4681 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4741 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4801 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4861 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4921 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4981 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5041 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5101 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5161 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5221 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5281 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5341 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5401 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5461 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5521 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5581 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5641 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5701 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5761 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5821 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5881 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5941 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6001 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6061 c
//
