LOCUS       sequence-331909-        5700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion pf NLS-mEos3.2-H-NSdbd-NLS into the chromosome of 
            a eukaryotic cell line carrying the Flp-IN/T-Rex system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5700)
  AUTHORS   Rashid FM, Mahlandt E, van der Vaart M, Boer DEC, Varela Alvarez M, 
            Henneman B, Brocken DJW, Voskamp P, Blok AJ, Shimizu TS, Meijer AH, 
            Luijsterburg MS, Goedhart J, Cremazy FGE, Dame RT
  TITLE     HI-NESS: a family of genetically encoded DNA labels based on a 
            bacterial nucleoid-associated protein.
  JOURNAL   Nucleic Acids Res. 2021 Nov 4. pii: 6414584. doi: 
            10.1093/nar/gkab993.
  PUBMED    34734265
REFERENCE   2  (bases 1 to 5700)
  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"; journalName: "Nucleic 
            Acids Res. 2021 Nov 4. pii: 6414584. doi: 10.1093/nar/gkab993."
COMMENT     Sequence Label: pRD437
FEATURES             Location/Qualifiers
     source          1..5700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    824..842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    845..863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     873..897
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             996..1016
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1020..1697
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     CDS             1872..1892
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2041..2058)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2047..2271
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2404..2423)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2555..2602
                     /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             2646..3242
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     3139..3159
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3378..3498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3414..3433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3468..3487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3547..3563)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3547..3563)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3560..3582)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3571..3587
                     /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(3595..3601,3602..3619,3620..3625))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3640..3661
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3778..3795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3949..4537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4029..4048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4708..5499,5500..5568))
                     /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     5331..5350
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5569..5673)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgtctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga
      841 gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc
      901 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
      961 cggtgtaacg gctagcgcta ccggtcgcca ccatgccaaa gaagaagagg aaggtaggca
     1021 tgagtgcgat taagccagac atgaagatca aactccgtat ggaaggcaac gtaaacgggc
     1081 accactttgt gatcgacgga gatggtacag gcaagccttt tgagggaaaa cagagtatgg
     1141 atcttgaagt caaagagggc ggacctctgc cttttgcctt tgatatcctg accactgcat
     1201 tccattacgg caacagggta ttcgccaaat atccagacaa catacaagac tattttaagc
     1261 agtcgtttcc taaggggtat tcgtgggaac gaagcttgac tttcgaagac gggggcattt
     1321 gcaacgccag aaacgacata acaatggaag gggacacttt ctataataaa gttcgatttt
     1381 atggtaccaa ctttcccgcc aatggtccag ttatgcagaa gaagacgctg aaatgggagc
     1441 cctccactga gaaaatgtat gtgcgtgatg gagtgctgac gggtgatatt gagatggctt
     1501 tgttgcttga aggaaatgcc cattaccgat gtgacttcag aactacttac aaagctaagg
     1561 agaagggtgt caagttacca ggcgcccact ttgtggacca ctgcattgag attttaagcc
     1621 atgacaaaga ttacaacaag gttaagctgt atgagcatgc tgttgctcat tctggattgc
     1681 ctgacaatgc aagacgagct gccgttaaat ctggcaccaa agctaaacgt gctcagcgtc
     1741 cggcaaaata tagctacgtt gacgaaaacg gcgaaactaa aacctggact ggccaaggcc
     1801 gtactccagc tgtaatcaaa aaagcaatgg atgagcaagg taaatccctc gacgatttcc
     1861 tgatcaagca accgaaaaag aaacgcaaag ttgggtaact gatcataatc agccatacca
     1921 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1981 ataaaatgaa tgcaattgtt gttgttaacg ctatccggcc cgtttaaacc cgctgatcag
     2041 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2101 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2161 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2221 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2281 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2341 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2401 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2461 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2521 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     2581 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtgctggc
     2641 gcgccaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     2701 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     2761 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     2821 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     2881 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     2941 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     3001 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     3061 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     3121 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     3181 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     3241 gacctaggca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3301 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3361 agttcttcgc ccaccccaac ttgtttattg cagcttaaat ggttacaaat aaagcaatag
     3421 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3481 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     3541 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3601 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3661 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3721 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3781 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3841 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3901 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3961 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4021 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4081 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4141 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4201 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4261 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4321 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4381 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4441 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4501 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4561 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4621 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4681 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4741 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4801 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4861 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4921 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4981 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5041 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5101 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5161 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5221 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5281 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5341 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5401 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5461 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5521 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5581 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5641 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
//
