LOCUS       sequence-329799-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of M-MLV reverse transcriptase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   Graham TGW, Dugast-Darzacq C, Dailey GM, Nguyenla XH, Van Dis E, 
            Esbin MN, Abidi A, Stanley SA, Darzacq X, Tjian R
  TITLE     Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 
            detection.
  JOURNAL   PLoS One. 2021 Feb 3;16(2):e0246647. doi: 
            10.1371/journal.pone.0246647. eCollection 2021.
  PUBMED    33534838
REFERENCE   2  (bases 1 to 7302)
  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: "PLoS One.";
            date: "2021-02-3"; volume: "16(2):e0246647. doi"; pages: " 
            10.1371/journal.pone.0246647. eCollection 2021"
COMMENT     Sequence Label: pET-28a_6H-MMLV_RT_D524N-6H
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1497..1514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1524..1541
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3564..3581
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3634..3652)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3648..3695
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3732..4187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3819..3838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4029..4050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4279..5094)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4279..5094)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5002..5021
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5086..5105
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5216..5804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5705..5724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5958..5975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5990..6129
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6055..6074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6215..6237
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6231..6422)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7231..7302,1..1011))
                     /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"
ORIGIN
        1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     1501 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatgacc ctaaatatag
     1561 aagatgagta tcggctacat gagacctcaa aagagccaga tgtttctcta gggtccacat
     1621 ggctgtctga ttttcctcag gcctgggcgg aaaccggggg catgggactg gcagttcgcc
     1681 aagctcctct gatcatacct ctgaaagcaa cctctacccc cgtgtccata aaacaatacc
     1741 ccatgtcaca agaagccaga ctggggatca agccccacat acagagactg ttggaccagg
     1801 gaatactggt accctgccag tccccctgga acacgcccct gctacccgtt aagaaaccag
     1861 ggactaatga ttataggcct gtccaggatc tgagagaagt caacaagcgg gtggaagaca
     1921 tccaccccac cgtgcccaac ccttacaacc tcttgagcgg gctcccaccg tcccaccagt
     1981 ggtacactgt gcttgattta aaggatgcct ttttctgcct gagactccac cccaccagtc
     2041 agcctctctt cgcctttgag tggagagatc cagagatggg aatctcagga caattgacct
     2101 ggaccagact cccacagggt ttcaaaaaca gtcccaccct gtttgatgag gcactgcaca
     2161 gagacctagc agacttccgg atccagcacc cagacttgat cctgctacag tacgtggatg
     2221 acttactgct ggccgccact tctgagctag actgccaaca aggtactcgg gccctgttac
     2281 aaaccctagg gaacctcggg tatcgggcct cggccaagaa agcccaaatt tgccagaaac
     2341 aggtcaagta tctggggtat cttctaaaag agggtcagag atggctgact gaggccagaa
     2401 aagagactgt gatggggcag cctactccga agacccctcg acaactaagg gagttcctag
     2461 ggacggcagg cttctgtcgc ctctggatcc ctgggtttgc agaaatggca gcccccttgt
     2521 accctctcac caaaacgggg actctgttta attggggccc agaccaacaa aaggcctatc
     2581 aagaaatcaa gcaagctctt ctaactgccc cagccctggg gttgccagat ttgactaagc
     2641 cctttgaact ctttgtcgac gagaagcagg gctacgccaa aggtgtccta acgcaaaaac
     2701 tgggaccttg gcgtcggccg gtggcctacc tgtccaaaaa gctagaccca gtagcagctg
     2761 ggtggccccc ttgcctacgg atggtagcag ccattgccgt actgacaaag gatgcaggca
     2821 agctaaccat gggacagcca ctagtcattc tggcccccca tgcagtagag gcactagtca
     2881 aacaaccccc cgaccgctgg ctttccaacg cccggatgac tcactatcag gccttgcttt
     2941 tggacacgga ccgggtccag ttcggaccgg tggtagccct gaacccggct acgctgctcc
     3001 cactgcctga ggaagggctg caacacaact gccttgatat cctggccgaa gcccacggaa
     3061 cccgacccga cctaacggac cagccgctcc cagacgccga ccacacctgg tatacgaatg
     3121 gaagcagtct cttacaagag ggacagcgta aggcgggagc tgcggtgacc accgagaccg
     3181 aggtaatctg ggctaaagcc ctgccagccg ggacatccgc tcagcgggct gaactgatag
     3241 cactcaccca ggccctaaag atggcagaag gtaagaagct aaatgtttat actgatagcc
     3301 gttatgcttt tgctactgcc catatccatg gagaaatata cagaaggcgt gggttgctca
     3361 catcagaagg caaagagatc aaaaataaag acgagatctt ggccctacta aaagccctct
     3421 ttctgcccaa aagacttagc ataatccatt gtccaggaca tcaaaaggga cacagcgccg
     3481 aggctagagg caaccggatg gctgaccaag cggcccgaaa ggcagccatc acagagactc
     3541 cagacacctc taccctcctc gagcaccacc accaccacca ctgagatccg gctgctaaca
     3601 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     3661 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3721 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4141 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4261 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4321 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4381 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4441 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4501 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     4561 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4621 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     4681 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4741 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4801 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4861 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4921 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4981 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5041 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     5101 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     5161 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5221 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5281 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5341 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     5401 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5461 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5521 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5581 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5641 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5701 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5761 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5821 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5881 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5941 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     6001 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     6061 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     6121 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     6181 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6241 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     6301 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     6361 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     6421 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     6481 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     6541 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     6601 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     6661 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     6721 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     6781 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     6841 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     6901 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     6961 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     7021 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     7081 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     7141 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     7201 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     7261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga ga
//
