LOCUS       sequence-256454-        5701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5701)
  TITLE     Emory Custom Cloning Core Plasmids - Oskar Laur
REFERENCE   2  (bases 1 to 5701)
  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"
COMMENT     Sequence Label: pGEX-4T1 NLS-GFP
FEATURES             Location/Qualifiers
     source          1..5701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             754..774
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(784..786,787..789,790..1503)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(784..786,787..789,790..1500)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(829..850)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1090..1109)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1437..1458
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1569..1591)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1736..1754)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1822..1926
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1927..1995,1996..2787)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2145..2164)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2958..3546
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3447..3466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3700..3717
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3790..3867
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3868..4950
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3887..3906)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4999..5004,5005..5022,5023..5029)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5037..5053
                     /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     5061..5077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5061..5077
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5073..5600
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5093..5110)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5093..5109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5102..5124)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tccccaaaaa agaagagaaa ggtagaagac
      781 cccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      841 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      901 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      961 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1021 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1081 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1141 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1201 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1261 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1321 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1381 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1441 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1501 tagctcgagc ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac
     1561 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     1621 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     1681 gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc
     1741 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     1801 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1861 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1921 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1981 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2041 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2101 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2161 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2221 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2281 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2341 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2401 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2461 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2521 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2581 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2641 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2701 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2761 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2821 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2881 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2941 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3001 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3061 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3121 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3181 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3241 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3301 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3361 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3421 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3481 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3541 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3601 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3661 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3721 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3781 ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     3841 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     3901 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     3961 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4021 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4081 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4141 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4201 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4261 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4321 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4381 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4441 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4501 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4561 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4621 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4681 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     4741 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     4801 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     4861 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4921 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     4981 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5041 gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg
     5101 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5161 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5221 aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg
     5281 tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa
     5341 actggcagat gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg
     5401 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
     5461 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta
     5521 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     5581 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     5641 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     5701 g
//
