LOCUS       sequence-494251-        5012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A GreenGate entry vector containing SV40 NLS and Rex NLS followed by
            a Hygromycin resistance expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5012)
  AUTHORS   Lu J, Jiang D
  TITLE     GreenGate vectors for CRISPR/dCas9-based epigenetic modifiers 
            targeting system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5012)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGGF-NLS-NOS terminator-Hygromycin resistance
FEATURES             Location/Qualifiers
     source          1..5012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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        2212..2230
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2212..2229
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2272..2292
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2323..2484
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             2512..2562
                     /codon_start=1
                     /product="nuclear localization signal from the human T-cell
                     leukemia virus type 1 (HTLV-1) Rex protein"
                     /label=Rex NLS
                     /translation="PKTRRRPRRSQRKRPPT"
     terminator      2595..2847
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        2919..3264
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3207..3227
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             3337..4362
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     polyA_signal    4403..4577
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(4598..4617)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4599..4617)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4843..4862)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4962..4984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgtagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtggctct cgaggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttaa taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacgagc tatttaggtg
     2221 acactataga agtgaagctt ggtctcaact accggcggtg gcggaggggc tccaaagaag
     2281 aagagaaagg tccgtacggg aggaggatct cggaccgaag agtacaagct tatcctgaac
     2341 ggtaaaaccc tgaaaggtga aaccaccacc gaagctgttg acgctgctac cgcggaaaaa
     2401 gttttcaaac agtacgctaa cgacaacggt gttgacggtg aatggaccta cgacgacgct
     2461 accaaaacct tcacggtaac cgaaggtggt ggtagcggtg gtggtactag tccaaaaaca
     2521 aggaggaggc cgcgaagatc acaacggaaa aggccgccta cgccatggcc gtaagtttga
     2581 gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
     2641 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
     2701 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
     2761 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
     2821 gcgcggtgtc atctatgtta cggtaccatg gtggagcacg acactctcgt ctactccaag
     2881 aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta
     2941 atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca
     3001 gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt
     3061 caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg
     3121 gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact
     3181 gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga
     3241 agttcatttc atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct
     3301 ctctcgagct ttcgcagatc cggggggcaa tgagatatga aaaagcctga actcaccgcg
     3361 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3421 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     3481 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     3541 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg agtttagcga gagcctgacc
     3601 tattgcatct cccgccgttc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3661 cccgctgttc tacaaccggt cgcggaggct atggatgcga tcgctgcggc cgatcttagc
     3721 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3781 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3841 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     3901 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     3961 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     4021 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     4081 ttcgagcgga ggcatccgga gcttgcagga tcgccacgac tccgggcgta tatgctccgc
     4141 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4201 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     4261 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     4321 ggaaaccgac gccccagcac tcgtccgagg gcaaagaaat agagtagatg ccgaccggga
     4381 tctgtcgatc gacaagctcg agtttctcca taataatgtg tgagtagttc ccagataagg
     4441 gaattagggt tcctataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg
     4501 tatttgtatt tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca
     4561 gtactaaaat ccagatcgta ttgagaccga attctcgccc tatagtgagt cgtattacct
     4621 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4681 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4741 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     4801 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     4861 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4921 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4981 gaggttttca ccgtcatcac cgaaacgcgc ga
//
