LOCUS       sequence-326595-        5527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion pf NLS-mEos3.2-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 5527)
  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 5527)
  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: pRD438
FEATURES             Location/Qualifiers
     source          1..5527
                     /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             992..1012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1016..1693
                     /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             1694..1714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1863..1880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1869..2093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2226..2245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2377..2424
                     /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             2468..3064
                     /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     2961..2981
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3200..3321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3237..3256)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3291..3310
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3370..3386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3370..3386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3383..3405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3394..3410
                     /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(3418..3424,3425..3442,3443..3448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3463..3484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3601..3618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3772..4360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3852..3871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4531..5322,5323..5391))
                     /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     5154..5173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5392..5496)
                     /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 agcctccggt
      961 gtaacggcta gcgctaccgg tcgccaccat gccaaagaag aagaggaagg taggcatgag
     1021 tgcgattaag ccagacatga agatcaaact ccgtatggaa ggcaacgtaa acgggcacca
     1081 ctttgtgatc gacggagatg gtacaggcaa gccttttgag ggaaaacaga gtatggatct
     1141 tgaagtcaaa gagggcggac ctctgccttt tgcctttgat atcctgacca ctgcattcca
     1201 ttacggcaac agggtattcg ccaaatatcc agacaacata caagactatt ttaagcagtc
     1261 gtttcctaag gggtattcgt gggaacgaag cttgactttc gaagacgggg gcatttgcaa
     1321 cgccagaaac gacataacaa tggaagggga cactttctat aataaagttc gattttatgg
     1381 taccaacttt cccgccaatg gtccagttat gcagaagaag acgctgaaat gggagccctc
     1441 cactgagaaa atgtatgtgc gtgatggagt gctgacgggt gatattgaga tggctttgtt
     1501 gcttgaagga aatgcccatt accgatgtga cttcagaact acttacaaag ctaaggagaa
     1561 gggtgtcaag ttaccaggcg cccactttgt ggaccactgc attgagattt taagccatga
     1621 caaagattac aacaaggtta agctgtatga gcatgctgtt gctcattctg gattgcctga
     1681 caatgccaga cgaccgaaaa agaaacgcaa agttgggtaa ctgatcataa tcagccatac
     1741 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     1801 acataaaatg aatgcaattg ttgttgttaa cgctatccgg cccgtttaaa cccgctgatc
     1861 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1921 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1981 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2041 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2101 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2161 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2221 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2281 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2341 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     2401 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtgctg
     2461 gcgcgccacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc
     2521 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc
     2581 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     2641 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     2701 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     2761 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     2821 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     2881 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     2941 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3001 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3061 ctgacctagg cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3121 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3181 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3241 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3301 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3361 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3421 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3481 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4381 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4441 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4501 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4561 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4621 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4681 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4741 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4801 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4861 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4921 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4981 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5041 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5101 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5161 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5221 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5281 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5341 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5401 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5461 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5521 tgacgtc
//
