LOCUS       sequence-462741-        7183 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of His-tagged GNL2. To be used as a probe..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7183)
  TITLE     Iuchi Lab GNL2 Plasmids 2025
REFERENCE   2  (bases 1 to 7183)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGNL2-#19
FEATURES             Location/Qualifiers
     source          1..7183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..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'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             921..932
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3141..3158
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3233..3255)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3400..3418)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3486..3590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3591..3659,3660..4451)
                     /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(3809..3828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4622..5210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5111..5130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5364..5381
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5454..5531
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5532..6614
                     /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(5551..5570)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6663..6668,6669..6686,6687..6693)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6701..6717
                     /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     6725..6741
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6725..6741
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6737..7183,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6757..6774)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6757..6773)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6766..6788)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 atcgaaggtc gtgggatccc cgaattcatg gtgaagccca
      961 agtacaaagg acggagcacc atcaacccgt ccaaggccag cacaaaccca gatcgagtgc
     1021 agggagcagg aggccaaaac atgagggacc gggccaccat ccggcgcctg aatatgtata
     1081 ggcaaaagga gcgcaggaac agtcgtggta aaataattaa acccctgcaa tatcaatcaa
     1141 cggtggcttc tggcacagtg gcaagagtag agccaaatat taaatggttt ggaaacacac
     1201 gtgtgattaa gcagtcatca ttacaaaaat ttcaagagga aatggataca gttatgaagg
     1261 atccatacaa agttgtcatg aagcaaagca agttaccaat gtctcttctc catgatcgaa
     1321 tccggcctca taacttgaag gtgcacattc ttgatactga aagttttgaa actacatttg
     1381 gccctaagtc acagaggaaa cgaccaaact tatttgcaag tgatatgcag tctcttatcg
     1441 aaaatgctga aatgtccact gagagctatg accagggcaa ggatcgtgat ttggtaactg
     1501 aagacactgg tgtgagaaat gaagctcaag aagagatcta taaaaaggga cagtccaaaa
     1561 gaatatgggg tgagctctac aaggtgatag attcatcaga tgttgtagtt caagttcttg
     1621 atgctagaga tccaatgggt actcgttccc ctcacattga aacttacctg aagaaggaaa
     1681 aaccttggaa acacctcatt tttgtactta acaaatgtga ccttgttcca acctgggcaa
     1741 caaaacggtg ggttgctgtc ctctcccagg attatccaac acttgctttc catgcaagcc
     1801 ttactaaccc gtttggcaag ggagcattca ttcagcttct gcggcagttt ggaaagttgc
     1861 acactgacaa gaaacagatc agtgttgggt tcattggcta tccaaatgtt ggcaagagct
     1921 ctgtgataaa tacattgcgt tctaagaaag tttgcaacgt ggctcccatt gcaggtgaaa
     1981 caaaggtctg gcagtatatt actttgatgc gtcggatatt cctgattgac tgtccaggtg
     2041 tggtttaccc ctctgaggac tccgagacag acattgtgct aaaaggagtt gttcaagtag
     2101 aaaaaattaa gagtcctgaa gaccacattg gtgctgtact tgaacgagca aagccagaat
     2161 atatcagcaa aacatacaag attgattctt gggagaatgc tgaggacttt cttgagaagc
     2221 tcgctttccg gactgggaag ttactaaagg gtggagagcc cgacttgcag actgtgggta
     2281 agatggtcct caatgactgg cagaggggcc ggattccttt ctttgtcaag ccacccaatg
     2341 cagagccact tgtggccccc cagcttctac cctcctcatc tttggaagtt gtcccagaag
     2401 cagcccagaa caatccaggg gaggaagtca cagaaactgc aggtgaaggg tcagaatcca
     2461 tcattaagga agaaacagaa gagaacagtc actgtgatgc taacacagag atgcagcaga
     2521 ttctcacacg agttcggcag aactttggta aaatcaacgt ggtgcctcag ttttctgggg
     2581 atgacctggt tcctgtggag gtgtcagatc ttgaggaaga gcttgagagc ttttctgatg
     2641 aagaggagga ggaacaggag caacaaagag atgatgcgga agagtcttcc tcggagcctg
     2701 aggaggaaaa tgtgggaaac gacaccaaag ccgttattaa agcactggat gagaagattg
     2761 ccaaatatca gaagtttcta gacaaagcca aagccaaaaa gttttcagca gtcagaatat
     2821 ccaagggact gagtgaaaag atatttgcaa aacctgaaga acaaagaaaa acactggaag
     2881 aagatgtaga tgacagagca ccttccaaaa agggaaagaa gcggaaggca caaagggaag
     2941 aggaacagga acattcaaat aaagctccca gggcgcttac atcaaaagaa cggaggcgag
     3001 cagtacgaca gcaacggccg aaaaaagttg gtgtgcgcta ctatgaaaca cacaacgtga
     3061 aaaataggaa caggaacaaa aagaagacca atgactcaga gggacagaaa cacaaacgca
     3121 aaaaattcag acaaaagcag caccaccacc accaccactg accgcgactc gagcggccgc
     3181 atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3241 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3301 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3361 gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata cgcctatttt
     3421 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3481 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3541 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3601 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3661 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3721 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3781 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     3841 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3901 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3961 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4021 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4081 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     4141 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4201 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4261 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4321 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4381 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4441 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4501 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4741 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5281 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5341 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5401 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat tccgacacca
     5461 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     5521 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     5581 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     5641 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     5701 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     5761 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     5821 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     5881 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     5941 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     6001 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     6061 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     6121 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     6181 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     6241 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     6301 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     6361 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     6421 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     6481 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     6541 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6601 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccccag
     6661 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6721 cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt tacaacgtcg
     6781 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     6841 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     6901 gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct
     6961 ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg
     7021 ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt
     7081 tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct
     7141 acaggaaggc cagacgcgaa ttatttttga tggcgttgga att
//
