LOCUS       sequence-331923-        5730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the insertion of NLS-mNeonGreen-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 5730)
  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 5730)
  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: pRD444
FEATURES             Location/Qualifiers
     source          1..5730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(74..93)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        265..644
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        645..848
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     799..819
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    850..868
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    871..889
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     899..923
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1022..1042
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1046..1753
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1301..1310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1928..1948
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2097..2114)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2103..2327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2460..2479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2611..2658
                     /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             2702..3298
                     /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     3195..3215
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3434..3554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3470..3489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3524..3543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3603..3619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3603..3619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3616..3638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3627..3643
                     /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(3651..3657,3658..3675,3676..3681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3696..3717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3834..3851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4005..4593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4085..4104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4764..5555,5556..5624))
                     /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     5387..5406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5625..5729)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
       61 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      121 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      181 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      241 atgtacgggc cagatatacg cgttgacatt gattattgtc tagttattaa tagtaatcaa
      301 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      361 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      421 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      481 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      541 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      601 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      661 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      721 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      781 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      841 gcagagctct ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag
      901 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
      961 gaagacaccg ggaccgatcc agcctccggt gtaacggcta gcgctaccgg tcgccaccat
     1021 gccaaagaag aagaggaagg taggcatggt gagcaagggc gaggaggata acatggcctc
     1081 tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg actttgacat
     1141 ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc tgaagtccac
     1201 caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt atggcttcca
     1261 tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg tagatggctc
     1321 cggctaccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta ctgttaacta
     1381 ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg ggactggttt
     1441 ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt gcaggtcgaa
     1501 gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt acaccactgg
     1561 aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca agccaatggc
     1621 ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc tcaagcactc
     1681 caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga tgggcatgga
     1741 cgagctgtac aaggctgccg ttaaatctgg caccaaagct aaacgtgctc agcgtccggc
     1801 aaaatatagc tacgttgacg aaaacggcga aactaaaacc tggactggcc aaggccgtac
     1861 tccagctgta atcaaaaaag caatggatga gcaaggtaaa tccctcgacg atttcctgat
     1921 caagcaaccg aaaaagaaac gcaaagttgg gtaactgatc ataatcagcc ataccacatt
     1981 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     2041 aatgaatgca attgttgttg ttaacgctat ccggcccgtt taaacccgct gatcagcctc
     2101 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2161 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2221 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2281 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2341 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2401 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2461 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2521 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2581 acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct
     2641 agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt gctggcgcgc
     2701 caccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     2761 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     2821 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     2881 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     2941 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3001 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3061 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     3121 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3181 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3241 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacc
     3301 taggcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3361 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3421 cttcgcccac cccaacttgt ttattgcagc ttaaatggtt acaaataaag caatagcatc
     3481 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3541 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3601 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3661 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3721 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3781 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3841 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3901 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3961 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4021 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4081 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4141 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4201 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4261 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4321 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4381 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4441 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4501 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4561 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4621 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4681 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4741 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4801 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4861 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4921 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4981 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5041 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5101 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5161 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5221 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5281 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5341 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5401 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5461 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5521 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5581 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5641 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5701 tagaaaaata aacaaatagg ggttccgcgc
//
