LOCUS       sequence-398447-        5768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFPuv with T7 induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5768)
  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 5768)
  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-GFPuv
FEATURES             Location/Qualifiers
     source          1..5768
                     /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(72..74,75..77,78..791)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(105..133)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     728..748
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      803..850
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      894..1349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     1191..1212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1464..2255,2256..2324))
                     /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     2087..2106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2325..2417)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2498..3086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2987..3006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3240..3257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3272..3411
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3337..3356)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3497..3519
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3513..3704)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4276..5358)
                     /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     5320..5339
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5359..5436)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5642..5661
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5752..5768,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5752..5768,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatataca tatggctagc aaaggagaag aacttttcac tggagttgtc ccaattcttg
      121 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
      181 atgctacata cggaaagctt acccttaaat ttatttgcac tactggaaaa ctacctgttc
      241 catggccaac acttgtcact actttctctt atggtgttca atgcttttcc cgttatccgg
      301 atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac
      361 gcactatatc tttcaaagat gacgggaact acaagacgcg tgctgaagtc aagtttgaag
      421 gtgataccct tgttaatcgt atcgagttaa aaggtattga ttttaaagaa gatggaaaca
      481 ttctcggaca caaactcgag tacaactata actcacacaa tgtatacatc acggcagaca
      541 aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacattgaa gatggatccg
      601 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
      661 cagacaacca ttacctgtcg acacaatctg ccctttcgaa agatcccaac gaaaagcgtg
      721 accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgagc
      781 tctacaaata actgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      841 gggttttttg ctagcgaaag gaggagtcga ctatatccgg attggcgaat gggacgcgcc
      901 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      961 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1021 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1081 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1141 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1201 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1261 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1321 ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag attatcaaaa
     1381 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1441 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1501 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     1561 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     1621 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1681 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1741 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     1801 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     1861 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     1921 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     1981 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2041 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2101 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2161 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2221 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2281 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2341 tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2401 ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt tcgttccact
     2461 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2521 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2581 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2641 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2701 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2761 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2821 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2881 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2941 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3001 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3061 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3121 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3181 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3241 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacacatc
     3301 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3361 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3421 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3481 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3541 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3601 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3661 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3721 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3781 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3841 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3901 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3961 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4021 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4081 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4141 aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga gctgactggg
     4201 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4261 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4381 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4441 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4501 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     4561 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4621 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4681 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4741 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4801 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4861 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4921 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4981 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5041 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5101 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5161 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5221 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5281 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5341 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5401 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5461 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5521 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5581 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5641 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5701 cgatgcgtcc ggcgtagcct aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     5761 tcactata
//
