LOCUS       sequence-398444-        5791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses redox sensitive GFP (roGFP) with T7 induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5791)
  AUTHORS   Hennigan JN, Menacho-Melgar R, Sarkar P, Golovsky M, Lynch MD
  TITLE     
  JOURNAL   Metab Eng. 2024 Sep;85:116-130. doi: 10.1016/j.ymben.2024.07.012. 
            Epub 2024 Jul 24.
  PUBMED    39059674
REFERENCE   2  (bases 1 to 5791)
  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: "Metab 
            Eng."; date: "2024-09"; volume: "85:116-130. doi"; pages: " 
            10.1016/j.ymben.2024.07.012. Epub 2024 Jul 24"
COMMENT     Sequence Label: pETM6-Amp-T7-roGFP
FEATURES             Location/Qualifiers
     source          1..5791
                     /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             join(71..73,74..76,77..787)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(116..137)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(377..396)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     724..745
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             794..811
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      826..873
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      917..1372
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     1214..1235
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1487..2278,2279..2347))
                     /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     2110..2129
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2348..2440)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2521..3109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3010..3029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3263..3280
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3295..3434
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3360..3379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3520..3542
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3536..3727)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4299..5381)
                     /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     5343..5362
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5382..5459)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5665..5684
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5775..5791,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5775..5791,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatataca atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      121 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      181 tgccacctac ggcaagctga ccctgaagtt catctccacc accggcaagc tgcccgtgcc
      241 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
      301 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      361 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      421 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      481 cctggggcac aagctggagt acaactacaa ctgccacaac gtctatatca tggccgacaa
      541 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      601 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
      661 cgacaaccac tacctgagca cctgctccgc cctgagcaaa gaccccaacg agaagcgcga
      721 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
      781 gtacaagaga tctcatcacc atcaccatca ctaactgagc aataactagc ataacccctt
      841 ggggcctcta aacgggtctt gaggggtttt ttgctagcga aaggaggagt cgactatatc
      901 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
      961 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1021 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1081 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1141 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1201 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1261 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1321 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc
     1381 acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1441 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1501 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1561 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1621 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1681 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1741 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1801 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1861 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1921 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1981 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2041 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2101 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2161 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2221 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2281 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2341 tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg
     2401 agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc
     2461 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2521 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2581 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2641 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2701 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2761 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2821 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2881 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2941 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3001 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3061 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3121 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3181 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3241 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3301 gcggtatttt ctccttacac atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3361 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3421 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3481 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3541 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3601 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3661 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3721 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3781 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3841 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3901 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3961 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4021 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4081 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4141 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     4201 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4261 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4381 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4441 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4501 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4561 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4621 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4681 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4741 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4801 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4861 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4921 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4981 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5041 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5101 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5161 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5221 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5281 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5341 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5401 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5461 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5521 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5581 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5641 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5701 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag cctaggatcg agatcgatct
     5761 cgatcccgcg aaattaatac gactcactat a
//
