LOCUS       sequence-331915-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion pf NLS-mTurquoise2-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 5739)
  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 5739)
  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: pRD439
FEATURES             Location/Qualifiers
     source          1..5739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        241..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    826..844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    847..865
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     875..899
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             998..1018
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1022..1024,1025..1027,1028..1738)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1067..1088)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1328..1347)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1675..1696
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1913..1933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2082..2099)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2088..2312
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2445..2464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2596..2643
                     /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             2687..3283
                     /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     3180..3200
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3419..3539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3455..3474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3509..3528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3588..3604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3588..3604)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3601..3623)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3612..3628
                     /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(3636..3642,3643..3660,3661..3666))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3681..3702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3819..3836)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3990..4578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4070..4089)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4749..5540,5541..5609))
                     /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     5372..5391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5610..5714)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
       61 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      121 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      181 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      241 gacattgatt attgtctagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      841 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      901 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      961 tccggtgtaa cggctagcgc taccggtcgc caccatgcca aagaagaaga ggaaggtagg
     1021 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1081 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1141 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1201 cctcgtgacc accctgtcct ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa
     1261 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1321 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1381 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1441 caagctggag tacaactact ttagcgacaa cgtctatatc accgccgaca agcagaagaa
     1501 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc
     1561 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1621 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     1681 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggc
     1741 tgccgttaaa tctggcacca aagctaaacg tgctcagcgt ccggcaaaat atagctacgt
     1801 tgacgaaaac ggcgaaacta aaacctggac tggccaaggc cgtactccag ctgtaatcaa
     1861 aaaagcaatg gatgagcaag gtaaatccct cgacgatttc ctgatcaagc aaccgaaaaa
     1921 gaaacgcaaa gttgggtaac tgatcataat cagccatacc acatttgtag aggttttact
     1981 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2041 tgttgttaac gctatccggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     2101 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2161 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2221 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2281 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2341 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2401 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2461 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2521 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2581 tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     2641 ttccttggcc aaaaagcctg aactcaccgc gacgtgctgg cgcgccaccg agtacaagcc
     2701 cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc
     2761 gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt
     2821 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2881 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     2941 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3001 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3061 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3121 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3181 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3241 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacctaggc acgtactacg
     3301 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3361 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3421 cttgtttatt gcagcttaaa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3481 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3541 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     3601 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3661 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3721 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3781 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3841 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3901 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3961 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4021 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4081 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4141 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4201 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4261 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4321 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4381 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4441 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4501 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4561 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4621 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4681 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4741 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4801 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4861 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4921 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4981 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5041 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5101 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5161 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5221 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5281 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5341 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5401 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5461 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5521 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5581 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5641 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5701 ataggggttc cgcgcacatt tccccgaaaa gtgccacct
//
