LOCUS       Exported                6926 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses engineered RT-1306 with His-tag.
ACCESSION   .
VERSION     .
KEYWORDS    pET30-RT1306
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  AUTHORS   Zhou H, Rauch S, Dai Q, Cui X, Zhang Z, Nachtergaele S, Sepich C, He
            C, Dickinson B.C.
  TITLE     Evolution of a reverse transcriptase to map N1-methyladenosine in 
            human messenger RNA
  JOURNAL   Nature Methods, 37, 23 September 2019
REFERENCE   2  (bases 1 to 6926)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6926
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     248..267
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(287..364)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     570..589
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     677..696
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        677..695
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    696..720
                     /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             735..757
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             768..785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2533..2551)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2547..2594
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2631..3086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2718..2737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2928..2949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3178..3993)
                     /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     3901..3920
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3985..4004
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4115..4703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4604..4623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4857..4874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4889..5028
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4954..4973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5114..5136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5130..5321)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6130..286)
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMVEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
       61 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
      121 gttgttgtgc cacgcggttg ggaatgtaat tcagctccac catcgccgct tccacttttt
      181 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
      241 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
      301 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
      361 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcacagtagt
      421 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
      481 aacagtcccc cggccacggg gcctgccacc atacccacgc caaaacaagc gctcatgagc
      541 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      601 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
      661 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      721 cctctagaaa taattttgtt taactttaag aaggagatat acatatgcac catcatcatc
      781 atcatggtgg aagcggtatg ccgattagcc cgattgaaac cgttccggtt aaactgaaac
      841 ctggtatgga tggtccgaaa gttaaacagt ggcctctgac cgaagaaaaa atcaaagcac
      901 tggtggaaat ttgcaccgag atggaaaaag aaggcaaaat cagcaaaatt ggtccggaaa
      961 atccgtataa tacaccggtt tttgccatca agaaaaagga tagcaccaaa tggcgtaaac
     1021 tgtttgctgc gaaggaactg aataaacgca cccaggattt ttgggaagtt cagctgggta
     1081 ttccgcatcc ggcaggtctg aaaaaaaaga aaagcgttac agttctggat gtgggtgatg
     1141 catattttag cgttccgctg gatgaagatt ttcgtaaata taccgcattt acgatcccga
     1201 gcattaataa cgaaacaccg ggtattcgct atcagtataa tgttctgccg caaggttgga
     1261 aaggtagtcc ggcaattttt cagagcagca tgaccaaaat tctggaaccg tttcgtaaac
     1321 agaatccgga tattgtgatc taccagtata tggatgatct gtatgttggt agcgatctgg
     1381 aaattggtca gcatcgtaca aaaattgaag aactgcgtca gcatctgctg cgttggggtc
     1441 tgaccacacc ggataaaaaa catcagaaag aaccgccttt tctgtatctg ggttatgaac
     1501 tgcatccgga taaatggacc gttcagccga ttgtgctgcc ggaaaaagat agctggaccg
     1561 ttaatgatat tcagaaactg gttggcaaac tgaattgggc aagccagatc tatccgggta
     1621 ttaaagttcg tcagctgtgt aaactgctgc gtggtacaaa agcactgaca gaagttattc
     1681 cgctgacgga agaagcagaa ctggaactgg cagaaaatcg tgaaattctg aaagaacctg
     1741 tgcacggcgt ttattatgat ccgagcaaag atctgattgc cgagatccag aaacaaggtc
     1801 aaggtcagtg gacctatcag atttatcaag aaccgttcaa gaatctgaaa accggtaaat
     1861 atgcacgtat gcgtggtgca cataccaatg atgtgaaaca gctgactgaa gccgttcaga
     1921 aaatcacaac cgaaagcatt gttatttggg gtaaaacccc gaaattcaaa ctgccgattc
     1981 agaaagagac atgggaaact tggtggaccg aatattggca ggcaacctgg attcctgaat
     2041 gggaatttgt taatacccct ccgctggtga aactgtggta tcagttagaa aaagaaccga
     2101 ttgttggtgc cgaaaccttt tatgttgatg gtgcagcaaa tcgcgaaacc aaactgggta
     2161 aagcaggtta tgttaccaac cgtggccgcc agaaggtagt tactttaacg gacaccacga
     2221 atcagaaaac agaattacaa gcgatttact tggcgcttca agatagcggt ctggaagtaa
     2281 acatcgttac ggacagccag tacgcacttg ggattattca ggcccagccg gatcagtctg
     2341 aaagtgaact ggtaaatcaa attattgagc agctgattaa aaaggagaag gtgtatctgg
     2401 cttgggtccc ggctcataaa ggtattggag ggaacgagca agttgacaaa ctggtttctg
     2461 cgggtattcg caaggtcctt taagatccgg ctgctaacaa agcccgaaag gaagctgagt
     2521 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2581 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2641 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2701 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2761 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2821 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2881 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2941 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3001 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3061 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     3121 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     3181 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     3241 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     3301 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     3361 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     3421 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     3481 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     3541 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     3601 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     3661 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     3721 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     3781 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     3841 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     3901 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     3961 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     4021 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4081 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4141 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4201 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4261 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4321 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4381 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4441 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4501 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4561 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4621 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4681 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4741 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4801 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4861 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4921 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4981 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5041 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5101 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5161 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5221 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5281 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5341 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5401 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5461 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5521 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5581 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5641 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5701 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5761 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5821 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5881 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5941 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6001 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6061 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     6121 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6181 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     6241 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     6301 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6361 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     6421 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6481 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6541 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6601 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6661 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6721 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6781 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6841 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6901 gacgccgctt cgttctacca tcgaca
//
