LOCUS       sequence-153508-        5507 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Spinach2 under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5507)
  AUTHORS   Wang XC, Wilson SC, Hammond MC
  TITLE     Next-generation RNA-based fluorescent biosensors enable anaerobic 
            detection of cyclic di-GMP.
  JOURNAL   Nucleic Acids Res. 2016 Jul 5. pii: gkw580.
  PUBMED    27382070
REFERENCE   2  (bases 1 to 5507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2016 Jul 5. pii: gkw580."
COMMENT     Sequence Label: pET31b-T7-Spinach2
FEATURES             Location/Qualifiers
     source          1..5507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(94..141)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     137..155
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(208..225)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      complement(232..278)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(447..466)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(448..466)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(550..569)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        775..852
                     /gene="lacI"
                     /label=lacI promoter
     CDS             853..1935
                     /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(872..891)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2744..2935
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2929..2951)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3037..3179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3092..3111
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3194..3211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3445..3464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4118..4909,4910..4978))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4741..4760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4979..5083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5109..5507,1..57))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5246..5267)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5458..5477
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
       61 attcgccaat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga
      121 ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt
      181 tcgggctttg ttagcagccg gatctcaatg gtggtggtgg tggtgctcga gcaaaaaacc
      241 cctcaagacc cgtttagagg ccccaagggg ttatgctatg gcgcccgaac agggacttga
      301 accctggacc cgcggccgga tgtaactagt tacggagctc acactctact caacaagtag
      361 gctgccgaag cagcctactg gacccgtcct tcaccatttc attcagttac atccggccgc
      421 tgctctaccg actgagctat ccgggcccct atagtgagtc gtattaattt cgcgggatcg
      481 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
      541 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
      601 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
      661 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
      721 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
      781 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
      841 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
      901 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
      961 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1021 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1081 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     1141 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1201 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     1261 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     1321 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     1381 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     1441 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     1501 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     1561 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     1621 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     1681 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     1741 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     1801 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     1861 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     1921 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     1981 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     2041 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     2101 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     2161 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     2221 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     2281 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     2341 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     2401 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     2461 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     2521 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     2581 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     2641 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     2701 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     2761 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     2821 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     2881 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2941 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3001 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3061 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3121 caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     3181 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3241 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3301 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3361 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3421 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3481 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3541 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3601 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3661 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagctggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5101 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5161 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5221 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5281 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5341 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5401 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5461 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcgg
//
