LOCUS       sequence-376981-        6171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express nGFPMNase  protein in E coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6171)
  AUTHORS   Stefanis NC, Bresnick JN, Kerwin RW, Schofield WN, McAllister G
  TITLE     Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic 
            brain.
  JOURNAL   Brain Res Mol Brain Res. 1998 Jan;53(1-2):112-9. doi: 
            10.1016/s0169-328x(97)00285-4.
  PUBMED    9473618
REFERENCE   2  (bases 1 to 6171)
  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: "Brain Res 
            Mol Brain Res."; date: "1998-01"; volume: "53(1-2):112-9. doi"; 
            pages: " 10.1016/s0169-328x(97)00285-4"
COMMENT     Sequence Label: pET28a-nGFPMNase
FEATURES             Location/Qualifiers
     source          1..6171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..398
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(418..495)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     701..720
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     804..823
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        804..822
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    823..847
                     /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)."
     RBS             862..884
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             903..920
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             930..947
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             951..983
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             996..1337
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             1389..1835
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ"
     CDS             1839..1856
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1909..1927)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1923..1970
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2007..2462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2094..2113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2304..2325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2554..3369)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2554..3369)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3277..3296
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3361..3380
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3491..4079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3980..3999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4233..4250
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4265..4404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4330..4349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4490..4512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4506..4697)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5506..6171,1..417))
                     /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 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
       61 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
      121 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
      181 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
      241 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
      301 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
      361 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
      421 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
      481 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
      541 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
      601 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      661 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      721 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      781 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      841 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca
      901 gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat atggctagca
      961 tgactggtgg acagcaaatg ggtcgcatgg atcaagtcca actggtggag tctggtggcg
     1021 ctttggtgca gccaggtggc tctctgcgtt tgtcctgtgc cgcttctggc ttcccagtga
     1081 accgctattc catgcgctgg tatcgccagg ctccaggcaa agagcgtgag tgggtagccg
     1141 gtatgtccag cgcgggtgat cgtagctcct atgaagactc cgtgaagggc cgtttcacca
     1201 tcagccgtga cgatgcccgt aacacggtgt atctgcaaat gaacagcttg aaacctgaag
     1261 atacggccgt gtattactgt aatgtgaacg tgggcttcga gtattggggc caaggcaccc
     1321 aggtcaccgt ctccagcggc ccgggaggcc aaggcggtgg ttctgagggt ggtggctccg
     1381 gtggaggggc aacttcaact aaaaaattac ataaagaacc tgcgacttta attaaagcga
     1441 ttgatggtga tacggttaaa ttaatgtaca aaggtcaacc aatgacattc agactattat
     1501 tggtcgacac acctgaaaca aagcatccta aaaaaggtgt agagaaatat ggtcctgaag
     1561 caagtgcatt taccaaaaaa atggtagaaa atgcaaagaa aattgaagtc gaattcgaca
     1621 aaggtcaaag aactgataaa tatggacgtg ggctagcgta tatttatgct gatggaaaaa
     1681 tggtaaacga agctttagtt cgtcaaggct tggctaaagt tgcttatgtt tacaaaccta
     1741 acaatacaca tgaacaacat ttaagaaaaa gtgaagcaca agcgaaaaaa gagaaattaa
     1801 atatttggag cgaagacaac gctgattcag gtcaataaca ccaccaccac caccactgag
     1861 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1921 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1981 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2041 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2101 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2161 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2221 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2281 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2341 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2401 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2461 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2521 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     2581 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2641 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2701 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2761 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2821 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2881 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2941 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     3001 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     3061 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     3121 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3181 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3241 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3301 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     3361 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3421 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3481 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3541 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3601 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3661 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3721 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3781 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3841 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3901 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3961 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4021 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4081 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4141 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4201 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4261 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4321 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4381 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4441 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4501 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4561 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4621 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4681 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4741 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4801 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4861 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4921 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4981 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5041 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5101 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     5161 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     5221 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5281 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5341 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5401 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5461 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5521 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5581 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5641 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5701 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5761 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5821 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5881 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5941 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     6001 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     6061 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     6121 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc a
//
