LOCUS       sequence-361816-        6191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli RtcA in bacterial expression cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6191)
  TITLE     pET-53-RtcA
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6191)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-53-RtcA
FEATURES             Location/Qualifiers
     source          1..6191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(854..1309)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(991..1012)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1203..1222
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1346..1393)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1389..1407
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1434..1457)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    1465..1489
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2507..2531)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2535..2552)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2565..2587
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2602..2626
                     /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)."
     primer_bind     complement(2626..2645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2627..2645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2733..2752)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2958..3035
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3036..4118
                     /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     complement(3055..3074)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4690..4881
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4875..4897)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4983..5122
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5038..5057
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5137..5154)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5308..5896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5388..5407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5977..6069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6070..6138,6139..6191,1..739)
                     /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"
ORIGIN
        1 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgcc
      841 atcgtgccgc cagaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
      901 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      961 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     1021 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     1081 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     1141 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     1201 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     1261 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     1321 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     1381 ggggttatgc tagttattgc tcagcggtgg cagcagccta ggttaatcac ttacttttcg
     1441 aactgcgggt ggctccaagc ggaaaccact ttgtacaaga aagctgggtc ttcaatgctc
     1501 acccgcgtta cgccatctgt ttctatcaaa ctaaaccgca ccggcaagaa acgctccacc
     1561 accgcgatat tggtcagcag atggcatgag ggatgggcga ccgtaaattc ccccgcgccc
     1621 gccagcgcca tcggtagcac cagttggtca gcaagatatt cccccaccgc cgccgtgctt
     1681 gccaggtagc gtttcacctc tttcaccaac tgtgccgcaa ccacctcggc actgacgcgc
     1741 ttttcaccga cgacaaaaaa gcgttcggtg atattttcac tttcgacttc aagcgagacg
     1801 gtattacccg gcccctggtc gcgcggcagg ttatgaatat tctgttcatg cagggaaaaa
     1861 ctccccgcca gtgtagcgat ttcacgctca gcaacatggc gcggcacacc agctaataga
     1921 acttctccac gcatctgcac aatgttcccg cgctcgccaa gttgcaaggt gttaaacgat
     1981 gccacgggcg agacttccgt tgccaccaca ccgcctccgg cagggtaaaa accgtggcgt
     2041 aacagcgtgg tttgctgatg aattcctatt ttcgccagca gcggctccag cacccggcgg
     2101 ataaaatccg caggcggcgc cgacgggtta tcggtgccgc cgctcacttc aacacgcgaa
     2161 ggtccatcgg caaaccacag cgcgggcagc accgtttgca gcaccagcgt acaacttccg
     2221 gcgctaccga tagcaaagcg gtaatcgccg ccgcgcacgg tgccgggccg gaagagcaga
     2281 cgctgcgacc ccagctccgc gccttccacg gttgctccgc aaatttccgt cgccgccttt
     2341 accgcggtca gatgctggcg caacagcccc ggtttcgccc gcccggcacg aatgctggtg
     2401 atggtaaatg gctggccggt tatcatcgac aggctcagcg ccgagcgcag gatctgcccg
     2461 ccaccttcgc cctgtgcgcc atccagcgca atcatccttt tcatagagcc tgcttttttg
     2521 tacaaacttg tgacgtgatg gtggtggtga tgtgccatgg tatatctcct tcttaaagtt
     2581 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     2641 tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg
     2701 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     2761 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     2821 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     2881 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     2941 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     3001 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     3061 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     3121 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     3181 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     3241 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     3301 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     3361 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     3421 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     3481 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3541 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     3601 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     3661 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     3721 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     3781 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct
     3841 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     3901 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     3961 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     4021 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4081 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     4141 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     4201 agctccttcc ggtgggcgcg gggcatgact agcatgatcg tgctcctgtc gttgaggacc
     4261 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     4321 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
     4381 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     4441 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     4501 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     4561 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     4621 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     4681 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     4741 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     4801 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     4861 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4921 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4981 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     5041 catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt
     5101 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5161 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5221 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5281 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5341 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5401 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5461 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5521 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5581 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5641 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5701 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5761 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5821 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5881 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5941 cgaaaactca cgttaaggga ttttggtcat gacctatttg tttatttttc taaatacatt
     6001 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaatca tgattgaaaa
     6061 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6121 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6181 ttgggtgcac g
//
