LOCUS       pGEX-4T-1        5469 bp DNA     circular SYN 21-NOV-2022
DEFINITION  Bacterial vector for expressing fusion proteins with a thrombin 
            site. For other reading frames, use pGEX-4T-2 or pGEX-4T-3.
ACCESSION   U13853
VERSION     .
KEYWORDS    pGEX-4T-1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5469)
  AUTHORS   GE Healthcare
  TITLE     Direct Submission
  JOURNAL   Exported Dec 9, 2022 from SnapGene 6.1.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5469
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5' Sequencing Primer
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             936..938
                     /codon_start=1
                     /label=Start Codon
                     /translation="M"
     CDS             939..1460
                     /codon_start=1
                     /label=Synaptotagmin 1 sequence
                     /translation="TVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVI
                     LEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQI
                     QKVQVVVTVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEE
                     EVDAMLAVKK"
     misc_feature    939..1457
                     /label=C2AB domain
     misc_feature    939..980
                     /label=C2A
     CDS             1014..1457
                     /label=C2B
     misc_feature    1089..1121
                     /label=C2B-loop 1
     CDS             1164..1175
                     /codon_start=1
                     /label=Lysine patch
                     /translation="KKKK"
     misc_feature    1185..1205
                     /label=C2B-loop 2
     misc_feature    1281..1307
                     /label=C2B-loop 3
     misc_feature    1386..1391
                     /label=SNARE binding
     misc_feature    1471..1481
                     /label=stop codons
                     /note="stop codons in all three reading frames"
     primer_bind     complement(1519..1541)
                     /label=pGEX 3' Sequencing Primer
     promoter        1772..1876
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1877..2737
                     /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"
     rep_origin      2908..3496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3740..3817
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3818..4900
                     /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"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccatgac cgtggatttt ggccatgtga
      961 ccgaggagtg gcgtgatctc cagagcgctg agaaagaaga gcaagagaaa ctgggtgaca
     1021 tctgcttctc cctccgctac gtccctactg ccggcaaact gactgttgtc attctggaag
     1081 ccaagaacct gaagaagatg gatgtgggtg gcttatctga tccctacgtg aagattcacc
     1141 tgatgcagaa cggtaagagg ctgaagaaga aaaagacgac gattaagaag aacacactca
     1201 acccctacta caacgagtcc ttcagctttg aagttccgtt cgagcaaatc cagaaagtgc
     1261 aagtggtggt aactgttttg gactatgaca agattggcaa gaacgacgcc atcggcaaag
     1321 tcttcgttgg ttacaacagc actggggcgg agctgcgaca ctggtcagac atgctggcca
     1381 acccccggcg acccatcgca cagtggcaca ctctgcaggt agaggaggag gttgatgcca
     1441 tgctggctgt caagaagtaa gaattcatcg tgactgactg acgatctgcc tcgcgcgttt
     1501 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     1561 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     1621 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtataatt cttgaagacg
     1681 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     1741 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1801 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1861 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1921 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1981 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2041 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     2101 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2161 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2221 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2281 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2341 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2401 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2461 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2521 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2581 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2641 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2701 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2761 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2821 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2881 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2941 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3001 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3061 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3121 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3181 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3241 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3301 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3361 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3421 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3481 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3541 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3601 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3661 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3721 ttcacaccgc ataaattccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg
     3781 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     3841 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     3901 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     3961 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     4021 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     4081 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     4141 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     4201 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     4261 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     4321 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     4381 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     4441 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     4501 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     4561 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     4621 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     4681 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     4741 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     4801 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     4861 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     4921 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     4981 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat
     5041 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     5101 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     5161 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca
     5221 ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc
     5281 gtcccctcaa actggcagat gcacggttac gatgcgccca tctacaccaa cgtaacctat
     5341 cccattacgg tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc
     5401 acatttaatg ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc
     5461 gttggaatt
//
