LOCUS       sequence-331919-        5736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion of NLS-eYFP-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 5736)
  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 5736)
  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: pRD442
FEATURES             Location/Qualifiers
     source          1..5736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(67..86)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        258..637
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        638..841
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     792..812
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    843..861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    864..882
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     892..916
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1015..1035
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1039..1041,1042..1752)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGINFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEGGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1081..1102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1342..1361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1689..1710
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1927..1947
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2096..2113)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2102..2326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2459..2478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2610..2657
                     /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             2701..3297
                     /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     3194..3214
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3433..3553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3469..3488)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3523..3542
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3602..3618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3602..3618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3615..3637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3626..3642
                     /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(3650..3656,3657..3674,3675..3680))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3695..3716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3833..3850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4004..4592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4084..4103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4763..5554,5555..5623))
                     /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     5386..5405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5624..5728)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
       61 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
      121 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
      181 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
      241 ggccagatat acgcgttgac attgattatt gtctagttat taatagtaat caattacggg
      301 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      361 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      421 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      481 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      541 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      601 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      661 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      721 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      781 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      841 tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt
      901 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
      961 ccgggaccga tccagcctcc ggtgtaacgg ctagcgctac cggtcgccac catgccaaag
     1021 aagaagagga aggtaggcgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1081 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1141 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1201 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac
     1261 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1321 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1381 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcaacttcaa ggaggacggc
     1441 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1501 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgagggcggc
     1561 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1621 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     1681 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1741 gagctgtaca aggctgccgt taaatctggc accaaagcta aacgtgctca gcgtccggca
     1801 aaatatagct acgttgacga aaacggcgaa actaaaacct ggactggcca aggccgtact
     1861 ccagctgtaa tcaaaaaagc aatggatgag caaggtaaat ccctcgacga tttcctgatc
     1921 aagcaaccga aaaagaaacg caaagttggg taactgatca taatcagcca taccacattt
     1981 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2041 atgaatgcaa ttgttgttgt taacgctatc cggcccgttt aaacccgctg atcagcctcg
     2101 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2161 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2221 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2281 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2341 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2401 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2461 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2521 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2581 cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta
     2641 gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtg ctggcgcgcc
     2701 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     2761 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
     2821 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2881 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2941 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     3001 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     3061 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     3121 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     3181 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3241 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacct
     3301 aggcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3361 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3421 ttcgcccacc ccaacttgtt tattgcagct taaatggtta caaataaagc aatagcatca
     3481 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3541 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3601 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3661 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3721 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5701 agaaaaataa acaaataggg gttccgcgca catttc
//
