LOCUS       VR124_pETv2_10Hi        6986 bp DNA     circular SYN 13-FEB-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  AUTHORS   Jan Soroczynski
  TITLE     Direct Submission
  JOURNAL   Exported Feb 13, 2023 from SnapGene 6.2.1
            https://www.snapgene.com
COMMENT     In NEB T7 Express lysY/Iq strain (C3013)
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..23
                     /label=Consensus phi-10 (T7) promoter (T7pCONS)
                     /note="color: #ff99cc; direction: RIGHT"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    20..23
                     /label=Inserted
                     /note="color: #ccffcc"
     protein_bind    24..48
                     /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)."
                     /note="color: #31849b"
     RBS             63..85
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     misc_feature    73..85
                     /label=Shine-Dalgarno
                     /note="color: #ffff99; direction: RIGHT"
     misc_feature    86..100
                     /label=TIR-2 Sequence
                     /note="color: #ccffcc; direction: RIGHT"
     CDS             104..133
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /note="color: #ffffff"
                     /translation="HHHHHHHHHH"
     CDS             167..328
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /note="color: #33cccc"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             416..1840
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /note="color: #008080"
                     /translation="ITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSI
                     TISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRH
                     QVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGK
                     WLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLY
                     LYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAV
                     LAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQ
                     RMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPD
                     ECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSK
                     LDGFLAAKDLMAQGIKI"
     misc_feature    572..574
                     /label=E54K
                     /note="Classical hyperactive Tn5 mutation, promotes Mosaic 
                     EnD Sequence binding.  "
                     /note="color: #ff0000"
     misc_feature    578..580
                     /label=M56
                     /note="M56A is an identified mutant, often introduced in 
                     combination with E54K and L372P, for the reason of 
                     inhibiting downstream translation initiation of the Tnp 
                     (Tn5) inhibitor. 
                     https://doi.org/10.1128/jb.174.4.1229-1239.1992
                     "
                     /note="color: #ffcc00"
     misc_feature    1526..1528
                     /label=L372P
                     /note="Classical hyperactive Tnp mutation, L372P likely 
                     causes a break in an alpha-helix that normally positions 
                     W450 (Reznikoff 2008)."
                     /note="color: #ff0000"
     terminator      1931..1978
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
     rep_origin      2015..2470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(2563..3378)
                     /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"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3500..4088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    4274..4416
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(4518..4709)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5518..6600)
                     /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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6601..6678)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 taatacgact cactataggg agaggaattg tgagcggata acaattcccc tctagaaata
       61 attttgttta actttaagaa ggagagcagc tatgcagctt agccatcatc atcatcatca
      121 ccaccaccat catgccgtgg gcggatccgg tggttcaggc ggtagttata aattgatcct
      181 gaacggcaaa actttgaagg gcgaaacaac aacagaagct gttgacgctg ctaccgctga
      241 aaaagtattt aaacagtatg caaacgacaa cggtgttgat ggagaatgga catacgatga
      301 cgcaaccaaa accttcactg tcacagaagg tggtgggagc ggaggcggtg gcagcggtgg
      361 cggcggtagt ggcggtggcg gttcgggcgg cggtggttcg ggtggcggag gcagcattac
      421 cagtgcactg catcgtgcgg cggattgggc gaaaagcgtg ttttctagtg ctgcgctggg
      481 tgatccgcgt cgtaccgcgc gtctggtgaa tgttgcggcg caactggcca aatatagcgg
      541 caaaagcatt accattagca gcgaaggcag caaagccatg caggaaggcg cgtatcgttt
      601 tattcgtaat ccgaacgtga gcgcggaagc gattcgtaaa gcgggtgcca tgcagaccgt
      661 gaaactggcc caggaatttc cggaactgct ggcaattgaa gataccacct ctctgagcta
      721 tcgtcatcag gtggcggaag aactgggcaa actgggtagc attcaggata aaagccgtgg
      781 ttggtgggtg catagcgtgc tgctgctgga agcgaccacc tttcgtaccg tgggcctgct
      841 gcatcaagaa tggtggatgc gtccggatga tccggcggat gcggatgaaa aagaaagcgg
      901 caaatggctg gccgctgctg caacttcgcg tctgcgcatg ggcagcatga tgagcaacgt
      961 gattgcggtg tgcgatcgtg aagcggatat tcatgcgtat ctgcaagata aactggccca
     1021 taacgaacgt tttgtggtgc gtagcaaaca tccgcgtaaa gatgtggaaa gcggcctgta
     1081 tctgtatgat cacctgaaaa accagccgga actgggcggc tatcagatta gcattccgca
     1141 gaaaggcgtg gtggataaac gtggcaaacg taaaaaccgt ccggcgcgta aagcgagcct
     1201 gagcctgcgt agcggccgta ttaccctgaa acagggcaac attaccctga acgcggtgct
     1261 ggccgaagaa attaatccgc cgaaaggcga aaccccgctg aaatggctgc tgctgaccag
     1321 cgagccggtg gaaagtctgg cccaagcgct gcgtgtgatt gatatttata cccatcgttg
     1381 gcgcattgaa gaatttcaca aagcgtggaa aacgggtgcg ggtgcggaac gtcagcgtat
     1441 ggaagaaccg gataacctgg aacgtatggt gagcattctg agctttgtgg cggtgcgtct
     1501 gctgcaactg cgtgaatctt ttactccgcc gcaagcactg cgtgcgcagg gcctgctgaa
     1561 agaagcggaa cacgttgaaa gccagagcgc ggaaaccgtg ctgaccccgg atgaatgcca
     1621 actgctgggc tatctggata aaggcaaacg caaacgcaaa gaaaaagcgg gcagcctgca
     1681 atgggcgtat atggcgattg cgcgtctggg cggctttatg gatagcaaac gtaccggcat
     1741 tgcgagctgg ggtgcgctgt gggaaggttg ggaagcgctg caaagcaaac tggatggctt
     1801 tctggccgcg aaagacctga tggcgcaggg cattaaaatc taactcgagc accaccacca
     1861 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1921 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1981 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2041 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2101 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2161 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2221 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2281 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2341 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2401 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2461 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2521 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2581 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2641 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2701 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2761 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2821 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2881 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2941 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3001 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     3061 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3121 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3181 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3241 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3301 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3361 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3421 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4261 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4321 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4381 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4441 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4501 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4561 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4621 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4681 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4741 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4801 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4861 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4921 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4981 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5041 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5101 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5161 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5221 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5281 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5341 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5401 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5461 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5521 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5581 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5641 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5701 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5761 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     5821 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5881 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5941 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     6001 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     6061 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     6121 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     6181 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     6241 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6301 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6361 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6421 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6481 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6541 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6601 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6661 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6721 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6781 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6841 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     6901 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     6961 ggatcgagat ctcgatcccg cgaaat
//
