LOCUS       sequence-326597-        5728 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 5728)
  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 5728)
  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..5728
                     /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..1720
                     /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(1158..1176)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1426..1445
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1895..1915
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2064..2081)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2070..2294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2427..2446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2578..2625
                     /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             2669..3265
                     /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     3162..3182
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3401..3522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3438..3457)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3492..3511
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3571..3587)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3571..3587)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3584..3606)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3595..3611
                     /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(3619..3625,3626..3643,3644..3649))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3664..3685
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3802..3819)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3973..4561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4053..4072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4732..5523,5524..5592))
                     /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     5355..5374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5593..5697)
                     /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 taggcgtgag
     1021 caagggcgag gaggataaca tggctatcat caaggagttc atgcgcttca aggtgcacat
     1081 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1141 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     1201 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     1261 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1321 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     1381 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     1441 gaagactatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     1501 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     1561 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     1621 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     1681 gggccgccac tccaccggcg gcatggacga gctgtacaag gctgccgtta aatctggcac
     1741 caaagctaaa cgtgctcagc gtccggcaaa atatagctac gttgacgaaa acggcgaaac
     1801 taaaacctgg actggccaag gccgtactcc agctgtaatc aaaaaagcaa tggatgagca
     1861 aggtaaatcc ctcgacgatt tcctgatcaa gcaaccgaaa aagaaacgca aagttgggta
     1921 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     1981 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acgctatccg
     2041 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2101 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2161 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2221 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2281 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2401 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2521 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     2581 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     2641 tgaactcacc gcgacgtgct ggcgcgccac cgagtacaag cccacggtgc gcctcgccac
     2701 ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     2761 cacgcgccac accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     2821 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     2881 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2941 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3001 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     3061 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3121 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3181 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3241 catgacccgc aagcccggtg cctgacctag gcacgtacta cgagatttcg attccaccgc
     3301 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3361 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3421 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3481 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3541 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3601 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3661 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3781 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3841 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3901 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5581 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5701 atttccccga aaagtgccac ctgacgtc
//
