LOCUS       sequence-331930-        5718 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 5718)
  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 5718)
  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..5718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        261..640
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        641..844
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     795..815
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    846..864
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    867..885
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     895..919
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1018..1038
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1042..1737
                     /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(1175..1193)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1443..1462
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1912..1932
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2081..2098)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2087..2311
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2444..2463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2595..2642
                     /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             2686..3282
                     /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     3179..3199
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3418..3538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3454..3473)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3508..3527
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3587..3603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3587..3603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3600..3622)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3611..3627
                     /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(3635..3641,3642..3659,3660..3665))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3680..3701
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3818..3835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3989..4577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4069..4088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4748..5539,5540..5608))
                     /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     5371..5390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5609..5713)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
      121 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
      181 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
      241 acgggccaga tatacgcgtt gacattgatt attgtctagt tattaatagt aatcaattac
      301 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      361 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      421 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      481 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      541 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      601 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      661 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      721 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      781 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      841 agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg
      901 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
      961 acaccgggac cgatccagcc tccggtgtaa cggctagcgc taccggtcgc caccatgcca
     1021 aagaagaaga ggaaggtagg catggtgagc aagggcgagg cagtgatcaa ggagttcatg
     1081 cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     1141 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     1201 cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc
     1261 atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga gggcttcaag
     1321 tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc
     1381 ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt ccctcctgac
     1441 ggccccgtaa tgcagaagaa gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc
     1501 gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc
     1561 tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat gcccggcgcc
     1621 tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac cgtggtggaa
     1681 cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct gtacaaggct
     1741 gccgttaaat ctggcaccaa agctaaacgt gctcagcgtc cggcaaaata tagctacgtt
     1801 gacgaaaacg gcgaaactaa aacctggact ggccaaggcc gtactccagc tgtaatcaaa
     1861 aaagcaatgg atgagcaagg taaatccctc gacgatttcc tgatcaagca accgaaaaag
     1921 aaacgcaaag ttgggtaact gatcataatc agccatacca catttgtaga ggttttactt
     1981 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2041 gttgttaacg ctatccggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2101 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2161 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2221 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2281 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2341 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2401 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2461 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2521 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2581 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     2641 tccttggcca aaaagcctga actcaccgcg acgtgctggc gcgccaccga gtacaagccc
     2701 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
     2761 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
     2821 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     2881 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     2941 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     3001 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     3061 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     3121 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     3181 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     3241 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacctaggca cgtactacga
     3301 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3361 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3421 ttgtttattg cagcttaaat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3481 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3541 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3601 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3661 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3721 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3781 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3841 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3901 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3961 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4021 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4081 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4141 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4201 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4261 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4321 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4381 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4441 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4501 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4561 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4621 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4681 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4741 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4801 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4861 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4921 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4981 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5041 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5101 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5161 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5221 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5281 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5341 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5401 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5461 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5521 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5581 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5641 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5701 taggggttcc gcgcacat
//
