LOCUS       sequence-384873-        6159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for bacterial O-GlcNAcase BtGH84 fused to enhanced GFP and 
            NLSs for effective nuclear localization, in backbone for T3 In Vitro
            Transcription..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6159)
  AUTHORS   Formichetti S, Chitnavis U, Sadowska A, Liu N, Boskovic A, Boulard M
  TITLE     Perturbing Nuclear Glycosylation in the Mouse Preimplantation Embryo
            Slows Down Embryonic Growth
  JOURNAL   Dev Cell 10 Jan 2024
REFERENCE   2  (bases 1 to 6159)
  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: "Dev Cell 10
            Jan 2024"
COMMENT     Sequence Label: pRN3P-NLS-EGFP-Btgh-3NLS
FEATURES             Location/Qualifiers
     source          1..6159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..20
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           45..87
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      110..119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             125..145
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             170..880
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFNDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(197..225)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     820..840
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3044..3064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3068..3088
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3092..3112
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           3135..3260
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(3348..3367)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3349..3367)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3374..3391)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3374..3390)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3383..3405)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3532..3987)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3669..3690)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3881..3900
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4014..4118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4119..4187,4188..4979)
                     /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(4337..4356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5150..5738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5639..5658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5892..5909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6026..6047
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6062..6067,6068..6085,6086..6092)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6100..6116
                     /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)."
     primer_bind     6105..6127
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6124..6140
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6124..6140
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aaattaaccc tcactaaagg gaacaaaagc tggagctaag cttgcttgtt ctttttgcag
       61 aagctcagaa taaacgctca actttggcag atctgaattc ctgcagcccg ccaccatggc
      121 ctctcctaag aaaaagagga aagtggaagc atcaggcggg ctgcagaata gtaaaggaga
      181 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
      241 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
      301 atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcac
      361 ttatggtgtt caatgctttt caagataccc agatcatatg aagcggcacg acttcttcaa
      421 gagcgccatg cctgagggat acgtgcagga gaggaccatc ttcttcaatg acgacgggaa
      481 ctacaagaca cgtgctgaag tcaagtttga gggagacacc ctcgtcaaca ggatcgagct
      541 taagggaatc gatttcaagg aggacggaaa catcctcggc cacaagttgg aatacaacta
      601 caactcccac aacgtataca tcatggccga caagcaaaag aacggcatca aagccaactt
      661 caagacccgc cacaacatcg aagacggcgg cgtgcaactc gctgatcatt atcaacaaaa
      721 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc
      781 tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
      841 agctgctggg attacacatg gcatggatga actatacaaa ggcggacaga atgtttctct
      901 ccaacctcca cctcagcagc tgatagtgca gaacaagact attgacctcc cggcagtcta
      961 ccagttgaat ggaggcgagg aggctaaccc ccacgctgtc aaggtactta aggaattgtt
     1021 gtccggcaag cagagctcaa aaaaggggat gctgattagt atcggggaga aaggagataa
     1081 atcagtaaga aagtacagta ggcagatacc agaccacaaa gaaggttact acctctctgt
     1141 taacgagaag gagatagtgc tggcaggcaa cgacgagcgc ggaacgtatt acgccttgca
     1201 aacttttgct cagctcttga aggacgggaa gttgcccgag gtagagataa aagactaccc
     1261 cagtgtccgg tataggggcg tggtagaggg gttctacggc actccatggt ctcatcaggc
     1321 tagactgagt cagttgaagt tctatggcaa aaacaagatg aacacctata tatatggtcc
     1381 caaggacgac ccgtatcact ccgctccaaa ctggcggctc ccctatcctg acaaggaggc
     1441 tgctcagctg caggagcttg tggctgttgc taacgaaaac gaggtcgatt ttgtatgggc
     1501 aattcatccc gggcaagata tcaagtggaa caaggaggat cgggatctgc tcctggccaa
     1561 gttcgaaaag atgtatcaac tgggggtccg atctttcgcc gtgttctttg acgatatcag
     1621 cggggaagga actaaccctc agaagcaggc agagttgctt aactatatcg atgagaagtt
     1681 tgcacaggtg aagcctgata ttaaccagct cgtgatgtgt cccacagagt ataacaagag
     1741 ctggagtaac cccaatggaa actacctcac tacacttggt gacaagctga acccctctat
     1801 ccagatcatg tggacagggg accgggttat atctgacata accagggacg gaatctcttg
     1861 gattaatgaa aggatcaagc gaccagccta catctggtgg aacttcccag tatctgatta
     1921 tgtcagagac cacctgctgc tggggcctgt gtacggcaat gatactacga tcgccaagga
     1981 aatgtccggc tttgtcacaa atcccatgga acacgctgag tcctctaaaa ttgctatcta
     2041 cagcgtggca tcatatgcct ggaatcccgc caagtatgac acctggcaga cttggaagga
     2101 tgcaatccgc acgatcctgc caagtgctgc agaggagctc gagtgtttcg ccatgcataa
     2161 cagcgacctg gggcccaatg gacatgggta tagacgagaa gaatcaatgg atatccagcc
     2221 tgccgcagag cggtttctga aagctttcaa agaaggaaaa aattacgaca aagccgattt
     2281 tgaaaccttg cagtacacct tcgagagaat gaaggagtct gccgacatcc tgctgatgaa
     2341 tacggaaaat aagccactga ttgtggagat cacaccttgg gtccaccaat tcaagttgac
     2401 cgctgaaatg ggcgaagagg ttctcaaaat ggtagagggg agaaacgaat cctacttctt
     2461 gcgcaagtac aaccatgtga aagctctcca acagcagatg ttctatattg accagacgtc
     2521 taaccagaac ccttatcaac caggtgtcaa gaccgccaca cgggtgataa agccactgat
     2581 agatcgcaca tttgcaacag ttgtgaaatt ttttaaccag aagttcaatg cccacctgga
     2641 cgccaccact gattacatgc cccacaagat gatcagcaac gtcgagcaaa ttaagaatct
     2701 ccccctgcag gtgaaagcaa atagggtcct gatctccccc gctaatgagg tggtcaagtg
     2761 ggccgctggt aattccgtgg aaatcgaact tgatgcaatc tatcctggtg agaatataca
     2821 gataaatttc ggcaaggacg caccctgcac ttggggccgc cttgagatca gcactgatgg
     2881 gaaagagtgg aaaaccgtgg acctgaagca gaaagagtcc cgcctttccg ctggactgca
     2941 gaaggctcca gtaaaattcg tccgattcac caacgtcagc gatgaggagc agcaggtcta
     3001 tttgcggcaa ttcgtcttga caattgagaa aaagactgga gaccccaaaa agaagcgcaa
     3061 agttgatcca aaaaaaaaaa ggaaagtcga tcctaagaag aaacggaaag tgtgagcgga
     3121 tctggttacc actaaaccag cctcaagaac acccgaatgg agtctctaag ctacataata
     3181 ccaacttaca ctttacaaaa tgttgtcccc caaaatgtag ccattcgtat ctgctcctaa
     3241 taaaaaraaa gtttcttcac attctaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaamc
     3301 cccccccccc cccccctgca gggccaagtc ggccggtacc caattcgccc tatagtgagt
     3361 cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     3421 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     3481 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg caaattgtaa
     3541 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3601 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3661 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3721 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3781 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3841 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3901 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3961 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4021 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4081 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4141 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4201 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4261 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4321 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4381 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4441 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4501 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4561 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4621 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4681 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4741 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4801 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4861 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4921 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4981 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5041 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5101 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5161 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5221 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5281 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5341 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5401 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5461 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5521 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5581 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5641 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5701 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5761 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5821 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5881 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5941 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     6001 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     6061 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     6121 acacaggaaa cagctatgac catgattacg ccaagctcg
//
