LOCUS       Exported                7306 bp ds-DNA     circular SYN 08-FEB-2021
DEFINITION  Bacterial vector for expression of N-terminally 6xHis-tagged 
            proteins with a thrombin site. For other reading frames, use 
            pET-28b(+) or pET-28c(+).
ACCESSION   .
VERSION     .
KEYWORDS    pET-28a_6H-MMLV_RT_D524N-6H
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   MilliporeSigma (Novagen)
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Feb 8, 2021 from SnapGene 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7306
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     source          5134..7146
                     /organism="Moloney murine leukemia virus"
                     /mol_type="genomic RNA"
                     /db_xref="taxon:11801"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /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"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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             5058..5063
                     /note="ribosome binding site"
     CDS             5071..5073
                     /codon_start=1
                     /product="start codon"
                     /label=ATG
                     /translation="M"
     CDS             5083..5100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5110..5127
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     gene            5134..7146
                     /locus_tag="MLVgp1"
                     /label=MLVgp1
                     /db_xref="GeneID:34950658"
     gene            5134..7146
                     /gene="gag-pol"
                     /locus_tag="MLVgp7"
                     /label=gag-pol
                     /db_xref="GeneID:2193424"
     mRNA            5134..7146
                     /gene="gag-pol"
                     /locus_tag="MLVgp7"
                     /label=gag-pol mRNA
                     /db_xref="GeneID:2193424"
     CDS             5134..7146
                     /codon_start=1
                     /gene="gag-pol"
                     /locus_tag="MLVgp7"
                     /product="Pr180"
                     /label=MMLV-RT
                     /note="Polyprotein; read-through stop codon;amber codon"
                     /db_xref="GeneID:2193424"
                     /experiment="experimental evidence, no additional 
                     detailsrecorded"
                     /protein_id="NP_057933.2"
                     /translation="TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQ
                     APLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKP
                     GTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
                     SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQY
                     VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT
                     EARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
                     QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL
                     DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMT
                     HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA
                     DHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGK
                     KLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHC
                     PGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL"
     gene            5134..7146
                     /gene="pol"
                     /locus_tag="MLVgp3"
                     /label=pol
                     /db_xref="GeneID:1491871"
     mat_peptide     5134..5136
                     /gene="gag-pol"
                     /locus_tag="MLVgp7"
                     /product="p14 PR"
                     /note="/protein_id=NP_955590.1"
     misc_difference 5155..5157
                     /label=H8Y
     primer_bind     5717..5736
                     /label=MLV RT_SEQfwd
     misc_feature    6703..6705
                     /label=D524N
     CDS             7150..7167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      7234..7281
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     5101 agcagcggcc tggtgccgcg cggcagccat atgaccctaa atatagaaga tgagtatcgg
     5161 ctacatgaga cctcaaaaga gccagatgtt tctctagggt ccacatggct gtctgatttt
     5221 cctcaggcct gggcggaaac cgggggcatg ggactggcag ttcgccaagc tcctctgatc
     5281 atacctctga aagcaacctc tacccccgtg tccataaaac aataccccat gtcacaagaa
     5341 gccagactgg ggatcaagcc ccacatacag agactgttgg accagggaat actggtaccc
     5401 tgccagtccc cctggaacac gcccctgcta cccgttaaga aaccagggac taatgattat
     5461 aggcctgtcc aggatctgag agaagtcaac aagcgggtgg aagacatcca ccccaccgtg
     5521 cccaaccctt acaacctctt gagcgggctc ccaccgtccc accagtggta cactgtgctt
     5581 gatttaaagg atgccttttt ctgcctgaga ctccacccca ccagtcagcc tctcttcgcc
     5641 tttgagtgga gagatccaga gatgggaatc tcaggacaat tgacctggac cagactccca
     5701 cagggtttca aaaacagtcc caccctgttt gatgaggcac tgcacagaga cctagcagac
     5761 ttccggatcc agcacccaga cttgatcctg ctacagtacg tggatgactt actgctggcc
     5821 gccacttctg agctagactg ccaacaaggt actcgggccc tgttacaaac cctagggaac
     5881 ctcgggtatc gggcctcggc caagaaagcc caaatttgcc agaaacaggt caagtatctg
     5941 gggtatcttc taaaagaggg tcagagatgg ctgactgagg ccagaaaaga gactgtgatg
     6001 gggcagccta ctccgaagac ccctcgacaa ctaagggagt tcctagggac ggcaggcttc
     6061 tgtcgcctct ggatccctgg gtttgcagaa atggcagccc ccttgtaccc tctcaccaaa
     6121 acggggactc tgtttaattg gggcccagac caacaaaagg cctatcaaga aatcaagcaa
     6181 gctcttctaa ctgccccagc cctggggttg ccagatttga ctaagccctt tgaactcttt
     6241 gtcgacgaga agcagggcta cgccaaaggt gtcctaacgc aaaaactggg accttggcgt
     6301 cggccggtgg cctacctgtc caaaaagcta gacccagtag cagctgggtg gcccccttgc
     6361 ctacggatgg tagcagccat tgccgtactg acaaaggatg caggcaagct aaccatggga
     6421 cagccactag tcattctggc cccccatgca gtagaggcac tagtcaaaca accccccgac
     6481 cgctggcttt ccaacgcccg gatgactcac tatcaggcct tgcttttgga cacggaccgg
     6541 gtccagttcg gaccggtggt agccctgaac ccggctacgc tgctcccact gcctgaggaa
     6601 gggctgcaac acaactgcct tgatatcctg gccgaagccc acggaacccg acccgaccta
     6661 acggaccagc cgctcccaga cgccgaccac acctggtata cgaatggaag cagtctctta
     6721 caagagggac agcgtaaggc gggagctgcg gtgaccaccg agaccgaggt aatctgggct
     6781 aaagccctgc cagccgggac atccgctcag cgggctgaac tgatagcact cacccaggcc
     6841 ctaaagatgg cagaaggtaa gaagctaaat gtttatactg atagccgtta tgcttttgct
     6901 actgcccata tccatggaga aatatacaga aggcgtgggt tgctcacatc agaaggcaaa
     6961 gagatcaaaa ataaagacga gatcttggcc ctactaaaag ccctctttct gcccaaaaga
     7021 cttagcataa tccattgtcc aggacatcaa aagggacaca gcgccgaggc tagaggcaac
     7081 cggatggctg accaagcggc ccgaaaggca gccatcacag agactccaga cacctctacc
     7141 ctcctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     7201 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     7261 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggat
//
