LOCUS       sequence-306277-        6926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of HIV reverse transcriptase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  TITLE     COVID-19 Diagnostic Toolkit Enzymes
REFERENCE   2  (bases 1 to 6926)
  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"
COMMENT     Sequence Label: pET30a_HIV-RT
FEATURES             Location/Qualifiers
     source          1..6926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     135..154
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        135..153
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    154..178
                     /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             193..215
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             226..243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1991..2009)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2005..2052
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2089..2544
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2176..2195)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2386..2407
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2636..3451)
                     /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(2636..3451)
                     /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     3359..3378
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3443..3462
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3573..4161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4062..4081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4315..4332
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4347..4486
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4412..4431)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4572..4594
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4588..4779)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5588..6670)
                     /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     6632..6651
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6671..6748)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
       61 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct
      121 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
      181 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat
      241 catggtggaa gcggtatgcc gattagcccg attgaaaccg ttccggttaa actgaaacct
      301 ggtatggatg gtccgaaagt taaacagtgg cctctgaccg aagaaaaaat caaagcactg
      361 gtggaaattt gcaccgagat ggaaaaagaa ggcaaaatca gcaaaattgg tccggaaaat
      421 ccgtataata caccggtttt tgccatcaag aaaaaggata gcaccaaatg gcgtaaactg
      481 gtggattttc gtgaactgaa taaacgcacc caggattttt gggaagttca gctgggtatt
      541 ccgcatccgg caggtctgaa aaaaaagaaa agcgttacag ttctggatgt gggtgatgca
      601 tattttagcg ttccgctgga tgaagatttt cgtaaatata ccgcatttac gatcccgagc
      661 attaataacg aaacaccggg tattcgctat cagtataatg ttctgccgca aggttggaaa
      721 ggtagtccgg caatttttca gagcagcatg accaaaattc tggaaccgtt tcgtaaacag
      781 aatccggata ttgtgatcta ccagtatatg gatgatctgt atgttggtag cgatctggaa
      841 attggtcagc atcgtacaaa aattgaagaa ctgcgtcagc atctgctgcg ttggggtctg
      901 accacaccgg ataaaaaaca tcagaaagaa ccgccttttc tgtggatggg ttatgaactg
      961 catccggata aatggaccgt tcagccgatt gtgctgccgg aaaaagatag ctggaccgtt
     1021 aatgatattc agaaactggt tggcaaactg aattgggcaa gccagatcta tccgggtatt
     1081 aaagttcgtc agctgtgtaa actgctgcgt ggtacaaaag cactgacaga agttattccg
     1141 ctgacggaag aagcagaact ggaactggca gaaaatcgtg aaattctgaa agaacctgtg
     1201 cacggcgttt attatgatcc gagcaaagat ctgattgccg agatccagaa acaaggtcaa
     1261 ggtcagtgga cctatcagat ttatcaagaa ccgttcaaga atctgaaaac cggtaaatat
     1321 gcacgtatgc gtggtgcaca taccaatgat gtgaaacagc tgactgaagc cgttcagaaa
     1381 atcacaaccg aaagcattgt tatttggggt aaaaccccga aattcaaact gccgattcag
     1441 aaagagacat gggaaacttg gtggaccgaa tattggcagg caacctggat tcctgaatgg
     1501 gaatttgtta atacccctcc gctggtgaaa ctgtggtatc agttagaaaa agaaccgatt
     1561 gttggtgccg aaacctttta tgttgatggt gcagcaaatc gcgaaaccaa actgggtaaa
     1621 gcaggttatg ttaccaaccg tggccgccag aaggtagtta ctttaacgga caccacgaat
     1681 cagaaaacag aattacaagc gatttacttg gcgcttcaag atagcggtct ggaagtaaac
     1741 atcgttacgg acagccagta cgcacttggg attattcagg cccagccgga tcagtctgaa
     1801 agtgaactgg taaatcaaat tattgagcag ctgattaaaa aggagaaggt gtatctggct
     1861 tgggtcccgg ctcataaagg tattggaggg aacgagcaag ttgacaaact ggtttctgcg
     1921 ggtattcgca aggtccttta agatccggct gctaacaaag cccgaaagga agctgagttg
     1981 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2041 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2101 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2161 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2221 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2281 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2341 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2401 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2461 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2521 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2581 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2641 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2701 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2761 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2821 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2881 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2941 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     3001 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     3061 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     3121 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     3181 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     3241 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     3301 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     3361 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     3421 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3481 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3541 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3601 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3661 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3721 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3781 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3841 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3901 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3961 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4021 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4081 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4141 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4201 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4261 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4321 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4381 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4441 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4501 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4561 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4621 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4681 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4741 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4801 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4861 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4921 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4981 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5041 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5101 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5161 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     5221 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     5281 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     5341 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     5401 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     5461 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5521 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5581 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5641 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5701 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5761 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5821 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     5881 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5941 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     6001 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     6061 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     6121 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     6181 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     6241 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6301 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6361 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6421 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6481 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6541 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6601 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6661 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6721 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6781 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6841 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6901 acgccgaaac aagcgctcat gagccc
//
