LOCUS       sequence-331916-        5727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion pf NLS-mCherry-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 5727)
  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 5727)
  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: pRD440
FEATURES             Location/Qualifiers
     source          1..5727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..54)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        226..605
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        606..809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     760..780
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    811..829
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    832..850
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     860..884
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             983..1003
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1007..1711
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1149..1167)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1417..1436
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1886..1906
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2055..2072)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2061..2285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2418..2437)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2569..2616
                     /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             2660..3256
                     /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     3153..3173
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3392..3512
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3428..3447)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3482..3501
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3561..3577)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3561..3577)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3574..3596)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3585..3601
                     /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(3609..3615,3616..3633,3634..3639))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3654..3675
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3792..3809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3963..4551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4043..4062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4722..5513,5514..5582))
                     /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     5345..5364
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5583..5687)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
       61 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
      121 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
      181 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattgt
      241 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      301 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      361 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      421 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      481 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      541 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      601 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      661 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      721 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      781 tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca
      841 gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
      901 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg tgtaacggct
      961 agcgctaccg gtcgccacca tgccaaagaa gaagaggaag gtaggcgtga gcaagggcga
     1021 ggaggataac atggctatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     1081 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1141 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     1201 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     1261 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1321 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     1381 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagactat
     1441 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     1501 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     1561 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     1621 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     1681 ctccaccggc ggcatggacg agctgtacaa ggctgccgtt aaatctggca ccaaagctaa
     1741 acgtgctcag cgtccggcaa aatatagcta cgttgacgaa aacggcgaaa ctaaaacctg
     1801 gactggccaa ggccgtactc cagctgtaat caaaaaagca atggatgagc aaggtaaatc
     1861 cctcgacgat ttcctgatca agcaaccgaa aaagaaacgc aaagttgggt aactgatcat
     1921 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     1981 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacgctatcc ggcccgttta
     2041 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2101 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2161 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2221 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2281 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2341 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2401 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2461 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2521 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     2581 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     2641 cgcgacgtgc tggcgcgcca ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     2701 cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     2761 caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     2821 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     2881 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     2941 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     3001 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     3061 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     3121 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     3181 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     3241 caagcccggt gcctgaccta ggcacgtact acgagatttc gattccaccg ccgccttcta
     3301 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3361 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt aaatggttac
     3421 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3481 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     3541 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3601 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3661 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3721 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3781 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3841 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3901 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3961 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4021 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4081 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4141 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4201 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4261 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4321 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4381 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4441 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4501 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4561 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4621 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4681 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4741 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4801 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4861 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4921 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4981 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5041 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5101 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5161 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5221 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5281 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5341 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5401 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5461 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5521 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5581 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5641 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5701 aaagtgccac ctgacgtcga cggatcg
//
