LOCUS       sequence-326600-        5719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion pf NLS-mScarlet-I-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 5719)
  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 5719)
  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: pRD443
FEATURES             Location/Qualifiers
     source          1..5719
                     /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..1711
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     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..3513
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3429..3448)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3483..3502
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3562..3578)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3562..3578)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3575..3597)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3586..3602
                     /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(3610..3616,3617..3634,3635..3640))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3655..3676
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3793..3810)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3964..4552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4044..4063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4723..5514,5515..5583))
                     /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     5346..5365
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5584..5688)
                     /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 taggcatggt
     1021 gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca tggagggctc
     1081 catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1141 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct
     1201 gtcccctcag ttcatgtacg gctccagggc cttcatcaag caccccgccg acatccccga
     1261 ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1321 cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa
     1381 ggtgaagctc cgcggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat
     1441 gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga agggcgacat
     1501 taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca agaccaccta
     1561 caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca agttggacat
     1621 cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg 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 ataatggtta
     3421 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3481 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3541 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3601 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3661 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3721 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3781 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3841 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3901 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3961 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4021 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4081 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4141 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4201 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4261 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4321 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4381 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4441 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4501 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4561 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4621 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4681 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4741 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4801 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4861 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4921 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4981 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5041 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5101 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5161 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5221 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5281 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5341 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5401 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5461 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5521 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5581 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5641 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5701 aaaagtgcca cctgacgtc
//
