LOCUS       sequence-331762-        5526 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 5526)
  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 5526)
  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..5526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(42..61)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        233..612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     767..787
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    818..836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    839..857
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     867..891
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             990..1010
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1014..1691
                     /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             1692..1712
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1861..1878)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1867..2091
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2224..2243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2375..2422
                     /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             2466..3062
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTSDVEVPEGPRTWCMTRKPGA"
     primer_bind     2959..2979
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3198..3318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3234..3253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3288..3307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3367..3383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3367..3383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3380..3402)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3391..3407
                     /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(3415..3421,3422..3439,3440..3445))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3460..3481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3598..3615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3769..4357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3849..3868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4528..5319,5320..5388))
                     /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     5151..5170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5389..5493)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
       61 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
      121 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      181 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      241 ttattgtcta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      301 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      421 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      781 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
      841 cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga
      901 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggtgt
      961 aacggctagc gctaccggtc gccaccatgc caaagaagaa gaggaaggta ggcatgagtg
     1021 cgattaagcc agacatgaag atcaaactcc gtatggaagg caacgtaaac gggcaccact
     1081 ttgtgatcga cggagatggt acaggcaagc cttttgaggg aaaacagagt atggatcttg
     1141 aagtcaaaga gggcggacct ctgccttttg cctttgatat cctgaccact gcattccatt
     1201 acggcaacag ggtattcgcc aaatatccag acaacataca agactatttt aagcagtcgt
     1261 ttcctaaggg gtattcgtgg gaacgaagct tgactttcga agacgggggc atttgcaacg
     1321 ccagaaacga cataacaatg gaaggggaca ctttctataa taaagttcga ttttatggta
     1381 ccaactttcc cgccaatggt ccagttatgc agaagaagac gctgaaatgg gagccctcca
     1441 ctgagaaaat gtatgtgcgt gatggagtgc tgacgggtga tattgagatg gctttgttgc
     1501 ttgaaggaaa tgcccattac cgatgtgact tcagaactac ttacaaagct aaggagaagg
     1561 gtgtcaagtt accaggcgcc cactttgtgg accactgcat tgagatttta agccatgaca
     1621 aagattacaa caaggttaag ctgtatgagc atgctgttgc tcattctgga ttgcctgaca
     1681 atgccagacg accgaaaaag aaacgcaaag ttgggtaact gatcataatc agccatacca
     1741 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1801 ataaaatgaa tgcaattgtt gttgttaacg ctatccggcc cgtttaaacc cgctgatcag
     1861 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1921 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1981 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2041 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2101 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2161 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2221 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2281 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2341 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     2401 ctctagaaag tataggaact tcctttgcca aaaagcctga actcaccgcg acgtgctggc
     2461 gcgccaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     2521 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     2581 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     2641 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     2701 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     2761 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     2821 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     2881 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     2941 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcacct
     3001 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     3061 gacctaggca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3121 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3181 agttcttcgc ccaccccaac ttgtttattg cagcttaaat ggttacaaat aaagcaatag
     3241 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3301 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     3361 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3421 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3481 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3541 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3601 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3661 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3721 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3781 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3841 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3901 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3961 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4021 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4081 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4141 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4201 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4261 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4321 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4381 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4441 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4501 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4561 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4621 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4681 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4741 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4801 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4861 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4921 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4981 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5041 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5101 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5161 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5221 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5281 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5341 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5401 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5461 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5521 cgtcga
//
