LOCUS       sequence-393468-        7234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  JZ73, bacterial expression construct expressing thermostabilised rat
            neurotensin receptor 1 (enNTS1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7234)
  AUTHORS   Bumbak F, Pons M, Inoue A, Paniagua JC, Yan F, Wu H, Robson SA, 
            Bathgate RAD, Scott DJ, Gooley PR, Ziarek JJ
  TITLE     Ligands selectively tune the local and global motions of neurotensin
            receptor 1 (NTS(1)).
  JOURNAL   Cell Rep. 2023 Jan 31;42(1):112015. doi: 
            10.1016/j.celrep.2023.112015. Epub 2023 Jan 20.
  PUBMED    36680775
REFERENCE   2  (bases 1 to 7234)
  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: "Cell Rep.";
            date: "2023-01-31"; volume: "42(1):112015. doi"; pages: " 
            10.1016/j.celrep.2023.112015. Epub 2023 Jan 20"
COMMENT     Sequence Label: pDS170-enNTS1
FEATURES             Location/Qualifiers
     source          1..7234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             202..231
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     regulatory      1186..1195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1303..1326
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1369..1392
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2527..2571
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2581..2604
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2629..3339
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVLCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAY
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLED
                     VTAAGITHGMDELYK"
     primer_bind     complement(2656..2684)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             3340..3369
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      3397..3491
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3598..3684
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3744..4826)
                     /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     4788..4807
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4827..4904)
                     /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."
     primer_bind     complement(4976..4998)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5084..5224
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5139..5158
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5239..5256)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5410..5998)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5490..5509)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6169..6960,6961..7029))
                     /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     6792..6811
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7030..7134)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7202..7220
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttctctagat aacgagggca aaccatgaaa
      121 ataaaaacag gtgcacgcat cctcgcatta tccgcattaa cgacgatgat gttttccgcc
      181 tcggctctcg ccaaactcga gcaccatcac catcaccatc accatcacca tgcgtcgacc
      241 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggcct cgctgaagtc
      301 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
      361 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
      421 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
      481 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
      541 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaagga tctgctgccg
      601 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
      661 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
      721 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
      781 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
      841 atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
      901 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
      961 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
     1021 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
     1081 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
     1141 ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac catggaaaac
     1201 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
     1261 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
     1321 gcgcagactg gatctaacaa caacaacaat aacaataaca acaacggtct cgaagtgctg
     1381 tttcagggcc cgggatccac ctctgaatct gacaccgcgg gtccgaactc tgacctggac
     1441 gtgaacaccg acatctactc taaagttctg gttaccgcga tttacctggc gctgtttgtt
     1501 gttggcaccg ttggtaacgg tgttaccctg ttcaccctgg cgcgtaaaaa atctctgcag
     1561 tctctgcagt ctcgtgttga ctactacctg ggttctctgg cgctgtcttc tctgctgatc
     1621 ctgctgtttg cgctgccggt tgatgtgtac aacttcatct gggttcacca cccgtgggcg
     1681 ttcggtgacg cgggttgcaa aggttactac ttcctgcgtg aagcgtgcac ctacgcgacc
     1741 gcgctgaacg ttgtttctct gtctgttgaa cgttacctgg cgatctgcca cccgttcaaa
     1801 gcgaaaaccc tgatgtctcg ttctcgtacc aaaaaattca tctctgcgat ctggctggcg
     1861 tctgcgctgc tgtctctgcc gatgctgttc acaatgggtc tgcagaacct gtctggtgac
     1921 ggcacccacc cgggtggtct ggtttgcacc ccgatcgttg acaccgcgac cctgcgtgtt
     1981 gttatccagc tgaacacctt catgtctttc ctgttcccga tgctggttgc gtctatcctg
     2041 aacaccgtta tcgcgcgtcg tctgaccgtt atggttcacc aggcggcgga acaggcgcgt
     2101 gtttccaccg ttggcaccca caacggtctg gaacactcta ccttcaacat gaccatcgaa
     2161 ccgggtcgtg ttcaggcgct gcgtcgtggt gttctggttc tgcgtgcggt tgttatcgcg
     2221 ttcgttgttt gctggctgcc gtaccacgtt cgtcgtctga tgttcgtgta catctctgac
     2281 gaacagtgga ccaccgcgct gttcgacttc taccactact tctacatgct gtctaacgcg
     2341 ctggtttacg tttctgcggc gatcaacccg atcctgtaca acctggtttc tgcgaacttc
     2401 cgtcaggttt tcctgtctac cctggcgagc ctgagtccgg gttggcgtca ccgtcgtaaa
     2461 aaacgtccga ccttctctcg taaaccgaac tctatgtctt ctaaccacgc gttctctacc
     2521 gctagcggtc tgaacgatat cttcgaagct cagaaaatcg aatggcacga aggctctgga
     2581 ctagaggtct tattccaagg tccaggcggg ggctcaggcg gtggctctag caaaggagaa
     2641 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
     2701 aaattttctg tccgtggaga gggtgaaggt gatgctacaa acggaaaact cacccttaaa
     2761 tttatttgca ctactggaaa actacctgtt ccgtggccaa cacttgtcac tactctgacc
     2821 tatggtgttc tatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag
     2881 agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggacc
     2941 tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta
     3001 aaaggtattg attttaaaga agatggaaac attctcggac acaaactgga gtacaacttt
     3061 aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa agcgtacttc
     3121 aaaattcgcc acaacgttga agatggttcc gttcaactag cagaccatta tcaacaaaat
     3181 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctctc gacacaatct
     3241 gttctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga ggatgtaact
     3301 gctgctggga ttacacatgg catggatgaa ctctacaaac accatcacca tcaccatcac
     3361 catcaccatt aataaaagct taattagctg agcttggact cctgttgata gatccagtaa
     3421 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     3481 ttggtgagaa tccaagctag tagtactgcg atgagtggca gggcggggcg taattttttt
     3541 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagttt gaggcatcaa
     3601 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3661 aacgctctcc tgagtaggac aaatccgccg ctctagagat ttccctcgac aattcgcgct
     3721 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3841 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3901 tgagagagtt gtagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3961 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4021 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4081 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4141 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4201 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4261 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4321 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4381 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4441 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4501 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4561 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4621 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4681 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4741 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4801 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4861 tatcatgcca taccgcgaaa ggttttgcac ctttcgatgg tgtcaacgta aatgcatgcc
     4921 gcttcgcctt ccctagctag agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     4981 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     5041 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     5101 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     5161 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5221 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5281 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5341 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5401 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5461 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5521 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5581 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5641 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5701 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5761 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5821 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5881 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5941 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6001 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6061 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6121 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6181 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6241 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6301 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6361 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6421 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6481 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6541 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6601 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6661 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6721 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6781 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6841 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6901 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6961 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7021 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7081 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7141 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     7201 aaataggcgt atcacgaggc cctttcgtct tcac
//
