LOCUS       sequence-141369-        7870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses the N-terminal fragment of manduca sexta soluble guanylyl 
            cyclase subunits alpha and beta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7870)
  AUTHORS   Hu X, Murata LB, Weichsel A, Brailey JL, Roberts SA, Nighorn A, 
            Montfort WR
  TITLE     Allostery in recombinant soluble guanylyl cyclase from Manduca 
            sexta.
  JOURNAL   J Biol Chem. 2008 Jul 25;283(30):20968-77. doi: 
            10.1074/jbc.M801501200. Epub 2008 May 30.
  PUBMED    18515359
REFERENCE   2  (bases 1 to 7870)
  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"; doi: 
            "10.1074/jbc.M801501200"; journalName: "J Biol Chem"; date: 
            "2008-07-25- 25"; volume: "283"; issue: "30"; pages: "20968-77"
COMMENT     Sequence Label: pETDuet-msGC-NT2
FEATURES             Location/Qualifiers
     source          1..7870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1459..1477
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1478..1502
                     /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             2816..2860
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2898..2916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2912..2959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2996..3451
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3083..3102)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3293..3314
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3566..4357,4358..4426))
                     /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     4189..4208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4427..4519)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4600..5188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5089..5108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5342..5359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5374..5516
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5442..5461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5602..5624
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5618..5809)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6381..7463)
                     /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     7425..7444
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7464..7541)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7747..7766
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7854..7870,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgctcactct
      121 taagcatatg agtgaggctt tgcaactcct gactgctcca tcgaatgaat gcctccatgc
      181 agctgtgacg tcactcacga agaatcaatc agatcactac cataaataca attgtttgcg
      241 tagattacca gacgacgtaa aaacttgtag aaattacgcg tacctacaag aaatttacga
      301 cgccgtgcga gcaactgaca gtgtcaacac gaaggatttt atggcaaaat taggggaata
      361 tttaatttta acagccttct cgcacaattg tcggttagag agagcgttta aatgtttagg
      421 cacaaattta acagaatttc tgaccacact agacagtgtt catgacgtgt tgcacgatca
      481 ggacacaccg cttaaagatg aaacgatgga gtacgaagcg aatttcgtat gcacgacctc
      541 gcaagaaggg aagatacagt tacacttgac cacggaaagc gaaccagtgg cgtacctttt
      601 agtaggcagt ctgaaagcca tagcgaaacg actgtatgat acacagacag acataagact
      661 gaggagctat accaatgatc ctaggaggtt taggtacgaa ataaatgcag tcccactaca
      721 ccagaaatca aaagaagata gctgcgaact tgtcaacgag gcagcttctg ttgccacctc
      781 gaccaaagtg acagatttga agatcggcgt ggctagcttc tgcaaagcgt ttccatggca
      841 tttcatcact gataaaaggc tggaactcgt tcaattggga gctggattca tgcgcctctt
      901 cggaacccac ttggcaacac acgggtcttc gctgggcacg tacttcagac tcctgcgtcc
      961 aagaggagtg ccgctggact ttcgcgagat cctcaagcgt gtcaacacac cgttcatgtt
     1021 ttgcctaaag atgcctggtt cgacagcact tgccgagggt ttggagatca aaggacaaat
     1081 ggtgttctgt gccgagtcag attctttgct gtttgtcggg tccccgttct tggacggtct
     1141 cgaaggtctg actggaaggg gactcttcat atccgatata cctcttcatg atgcgacgag
     1201 agatgtcatc ttagtcggag aacaggctag ggctcaggat ggtttgcgaa gacgaatgga
     1261 caagctgaag aactcaatag aagaagcgag caaagcagta gacaaggaac gagagaagaa
     1321 cgtcagcctg ctgcatttaa tattcccacc ccacatcgcc aaaaggcttt ggctaggcga
     1381 gtgatagaag cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca
     1441 cggccgcata atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1501 ccccatctta gtatattagt taagtataag aaggagatat acatatgtac gggtttgtga
     1561 actatgccct cgagctcctt gtgatgaaaa cttttgacga agaaacatgg gagacgataa
     1621 agaaaaaggc cgacgtggct atggaaggca gtttcctagt gagacagatt tatgaggatg
     1681 agatcactta taatttgatc accgctgccg ttgaggtctt gcaaataccc gctgacgcta
     1741 tattggagct atttggaaaa acgttcttcg agttttgcca agattccggg tatgataaaa
     1801 ttctgcaagt acttggagcc acgccacgag attttctaca gaatctggac ggcctgcacg
     1861 atcatctcgg gacattatat cctggcatga gatccccctc gttccgatgc accgagcgtc
     1921 ctgaagatgg cgcccttgtg cttcattatt actccgacag gcctggtctg gagcatatcg
     1981 ttattggaat tgtaaagact gtggcgagca aactccataa caccgaagtg aaggtggaaa
     2041 tcctaaagac caaagaggaa tgtgaccacg tccagttctt gatcactgaa acttccacca
     2101 ccggtagggt gtctgcacct gagatagcag agattgaaac gttgtctctt gaaccaaaag
     2161 tgagtccagc gacattctgc cgagtgtttc cattccattt gatgtttgat cgtgatctga
     2221 acatcgtcca agcgggacgt actgtgtctc gtctactccc aagagttacc agaccaggct
     2281 gtaagattac tgatgtcttg gatactgtgc gtccccactt agagatgacg tttgctaacg
     2341 tcctggctca tattaacacg gtctacgttt tgaaaaccaa acctgaagag atgagtgtca
     2401 ctgatcctca tgaggaaatc gcttctcttc gcttgaaagg tcaaatgctg tacattccag
     2461 aaacagacgt ggttgtattc cagtgttatc caagtgtgac caatttggac gatctgactc
     2521 gccgcggcct atgcattgct gacattccac ttcacgatgc gacacgagac ctggttctca
     2581 tgtccgagca atttgaagct gattataagc tgacccagaa tctggaagtt ttgactgaca
     2641 aacttcaaca aacattccga gagctggaat tggaaaaaca gaagacggac aggcttcttt
     2701 actcagtgct gcctataagc gtggccactg aacttcgtca ccgtcgctga aagggcgaat
     2761 tctgcagata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt ctggtaaaga
     2821 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     2881 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2941 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3001 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3061 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3121 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3181 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3241 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3301 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3361 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3421 aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg agattatcaa
     3481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3541 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3601 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3661 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3721 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3781 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3841 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3901 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3961 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4021 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4081 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4141 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4201 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4261 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4321 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4381 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4441 aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4501 atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt tttcgttcca
     4561 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4621 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4681 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4741 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4801 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4861 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4921 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4981 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5041 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5101 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5161 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5221 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5281 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5341 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5401 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     5461 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     5521 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5581 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5641 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     5701 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     5761 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     5821 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     5881 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     5941 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     6001 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     6061 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     6121 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     6181 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     6241 tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga
     6301 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     6361 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6421 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     6481 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     6541 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     6601 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     6661 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     6721 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     6781 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     6841 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     6901 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     6961 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     7021 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     7081 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     7141 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     7201 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     7261 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     7321 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     7381 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     7441 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     7501 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     7561 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     7621 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     7681 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     7741 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     7801 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg
     7861 actcactata
//
