LOCUS       sequence-476974-        6401 bp    DNA     circular SYN 24-JUN-2025
DEFINITION  E. coli expression vector for E. coli RtcB RNA ligase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6401)
  AUTHORS   Peach SE, York K, Hesselberth JR
  TITLE     Global analysis of RNA cleavage by 5'-hydroxyl RNA sequencing.
  JOURNAL   Nucleic Acids Res. 2015 May 22. pii: gkv536.
  PUBMED    26001965
REFERENCE   2  (bases 1 to 6401)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2015 May 22. pii: gkv536."
COMMENT     Sequence Label: pET-53-RtcB
FEATURES             Location/Qualifiers
     source          1..6401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             94..111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    115..139
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1367..1391)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1399..1422
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1449..1467)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1463..1510
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1547..2002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1634..1653)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1844..1865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2117..2908,2909..2977))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2740..2759
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2978..3070)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3151..3739
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3640..3659
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3893..3910
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3925..4064
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3990..4009)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4150..4172
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4166..4357)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4929..6011)
                     /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     5973..5992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6012..6089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6295..6314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gcacatcacc accaccatca cgtcacaagt
      121 ttgtacaaaa aagcaggctc tatgaattac gaattactga ccactgaaaa tgccccggta
      181 aaaatgtgga ccaaaggcgt gccggtagag gccgatgcgc gtcagcaact tattaatacg
      241 gcgaagatgc cgtttatttt caaacatatt gcggtaatgc ctgatgtaca cctgggtaaa
      301 ggttccacca ttggtagcgt gatcccgacc aaaggggcga ttattccggc ggcggtgggc
      361 gtggatattg gctgtggaat gaacgcgctg cgtaccgcgt taacggcgga agacctgcct
      421 gaaaacctgg cagagctgcg tcaggcgatt gaaacggccg tgccgcacgg gcgtaccact
      481 ggccgttgta aacgtgataa aggtgcctgg gaaaatccac ctgttaacgt cgatgctaaa
      541 tgggctgagc ttgaagccgg ttatcagtgg ttaacgcaaa aatatccccg tttcctgaat
      601 accaataact ataaacacct gggaacgctg ggaaccggta accactttat tgaaatctgc
      661 cttgatgagt cggaccaggt gtggattatg ctgcactccg gttcacgcgg aattggtaac
      721 gccatcggga cttactttat cgatctggca caaaaagaga tgcaggaaac gcttgagacg
      781 ttgccgtcgc gtgatctggc gtactttatg gaaggtacgg aatactttga tgattacctg
      841 aaagccgtgg cctgggcgca gctttttgcc agccttaacc gcgatgcgat gatggaaaac
      901 gtggtaacgg cattgcagag cattacgcag aaaacggtca gacagccaca aacgctggcg
      961 atggaagaga tcaactgtca ccacaactat gtgcaaaaag aacagcactt tggtgaagag
     1021 atctacgtga cgcgtaaagg cgcggtgtct gcgcgtgctg gtcaatatgg aattattccc
     1081 ggttcgatgg gagcaaaaag ctttatcgtc cgtgggctgg gaaatgaaga gtcgttctgt
     1141 tcgtgcagcc acggtgccgg gcgggtaatg agccgaacta aagcgaaaaa actgttcagc
     1201 gtggaagatc aaattcgtgc caccgcgcat gtggaatgcc gtaaagatgc cgaagtgatc
     1261 gacgaaatcc cgatggcgta taaagatatt gatgcggtga tggcggcaca aagcgatctg
     1321 gtggaagtta tctataccct gcgtcaggtg gtgtgcgtaa aaggagaccc agctttcttg
     1381 tacaaagtgg tttccgcttg gagccacccg cagttcgaaa agtaagtgat taacctaggc
     1441 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1501 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1561 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1621 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1681 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1741 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1801 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1861 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1921 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1981 aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct
     2041 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2101 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2161 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2221 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2281 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2341 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2401 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2461 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2521 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2581 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2641 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2701 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2761 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2821 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2881 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2941 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat
     3001 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3061 aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3121 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3181 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3241 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3301 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3361 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3421 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3481 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3541 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3601 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3661 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3721 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3781 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3841 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3901 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3961 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4021 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4081 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4141 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4201 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4261 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4321 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4381 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4441 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4501 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4561 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4621 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4681 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4741 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4801 ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4861 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4921 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4981 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5041 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5101 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5161 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     5221 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5281 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5341 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5401 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5461 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5521 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5581 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5641 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5701 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5761 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5821 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5881 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5941 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6001 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6061 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6121 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6181 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6241 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6301 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6361 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa t
//
