LOCUS       sequence-74443-e        6041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6041)
  AUTHORS   Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, 
            Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA
  TITLE     Design of orthogonal genetic switches based on a crosstalk map of 
            sigmas, anti-sigmas, and promoters.
  JOURNAL   Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58.
  PUBMED    24169405
REFERENCE   2  (bases 1 to 6041)
  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: "Mol Syst 
            Biol."; date: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pVRa16_3622
FEATURES             Location/Qualifiers
     source          1..6041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             616..633
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(686..704)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      700..747
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      784..1239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(871..890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1081..1102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1266..1370
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1371..1439,1440..2231)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1589..1608)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2402..2990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2891..2910
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3144..3161
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3176..3318
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3244..3263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3404..3426
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3420..3611)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4420..5502)
                     /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     5464..5483
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5503..5580)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5786..5805
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5889..5908
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5889..5907
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5908..5932
                     /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             5947..5969
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5988..6005
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6015..6038
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgcagcgta ccaatagcca ggatgttctg agcacccgtg aaagccagct gcaggcatta
       61 ctgctgcagg gtctggcagg cgataccttt gcatatcgtc agtttctgac cgcactggca
      121 gcacatattc gtggttttct gcgtcgtcgt ctgccgcagc atccggcaga agttgaagat
      181 ctgctgcaag aagttctgct ggcagttcat aatgcacgtc atacctatca ggcacgtcag
      241 ccgctgaccg catgggttca ggcaattgca cgttataaac tggcagatca tctgcgtagc
      301 catgcacgtc gtgaagcacg tcatgatctg ctggatgatg atagcgaact gtttgcagca
      361 agtgatgaac agcctgcaca ggcaagccgt gatctgggta aactgctggg tcagctgccg
      421 gatcgtcagc gtctgccgat tgttcatgtt aaactggaag gtctgagcgt tgaagaaacc
      481 gcacagatta ccggtctgag cagcagcgca gttaaagttg gtattcatcg tggtctgaaa
      541 gcactgggca aactgattcg tggtaaaggt catgatgaag atcgttaagg atccaagctt
      601 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      661 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      721 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      781 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      841 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      901 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      961 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1021 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1081 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1141 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1201 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc aggtggcact tttcggggaa
     1261 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1321 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1381 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1441 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     1501 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1561 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     1621 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     1681 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     1741 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     1801 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     1861 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     1921 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     1981 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2041 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2101 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2161 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2221 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2281 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2341 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2401 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2461 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2521 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2581 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2641 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2701 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2761 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2821 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2881 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2941 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3001 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3061 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3121 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3181 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct
     3241 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3301 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3361 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3421 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     3481 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     3541 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     3601 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     3661 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     3721 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     3781 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     3841 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     3901 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     3961 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4021 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4081 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4141 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4201 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4261 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4321 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4381 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4441 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4501 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     4561 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     4621 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     4681 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     4741 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     4801 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     4861 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     4921 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     4981 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5041 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5101 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5161 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5221 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5281 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5341 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     5401 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     5461 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     5521 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     5581 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     5641 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     5701 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     5761 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     5821 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     5881 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     5941 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
     6001 atcacagcag cggcctggaa gttctgttcc aggggcccca t
//
