LOCUS       sequence-210101-        3176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ColE1 BbsI cloning vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3176)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA
  TITLE     Engineered promoters enable constant gene expression at any copy 
            number in bacteria.
  JOURNAL   Nat Biotechnol. 2018 Mar 19. pii: nbt.4111. doi: 10.1038/nbt.4111.
  PUBMED    29553576
REFERENCE   2  (bases 1 to 3176)
  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: "Nat 
            Biotechnol. 2018 Mar 19. pii: nbt.4111. doi: 10.1038/nbt.4111."
COMMENT     Sequence Label: pTHSSe_52
FEATURES             Location/Qualifiers
     source          1..3176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..56
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     misc_RNA        64..113
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(156..158,159..872)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             156..869
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(186..214)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     809..829
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      889..960
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      976..1003
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1022..1042
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1043..1100
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(1124..1141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1272..1860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1761..1780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2055..2846,2847..2915))
                     /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     2678..2697
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2916..3020)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3088..3106
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3110..3153)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    join(3156..3176,1)
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 gggaggtgtc ttcggcgcgc ctttacggct agctcagtcc taggtactat gctagcaagg
       61 tagctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa cagcctctac
      121 aaataatttt gtttaatcac acaggaaagt actagatgag caaaggagaa gaacttttca
      181 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
      241 tccgtggaga gggtgaaggt gatgctacaa acggaaaact cacccttaaa tttatttgca
      301 ctactggaaa actacctgtt ccgtggccaa cacttgtcac tactctgacc tatggtgttc
      361 aatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag agtgccatgc
      421 ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggacc tacaagacgc
      481 gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aagggtattg
      541 attttaaaga agatggaaac attcttggac acaaactcga gtacaacttt aactcacaca
      601 atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc
      661 acaacgttga agatggttcc gttcaactag cagaccatta tcaacaaaat actccaattg
      721 gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct gtcctttcga
      781 aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga
      841 ttacacatgg catggatgag ctctacaaat aatactagag ccaggcatca aataaaacga
      901 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
      961 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatag aagacatcgc
     1021 ttactagtag cggccgctgc agtccggcaa aaaagggcaa ggtgtcacca ccctgccctt
     1081 tttctttaaa accgaaaaga ttacttcgcg ttatgcaggc ttcctcgctc actgactcgc
     1141 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaattcatg
     1201 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1261 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1321 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1381 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1441 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1501 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1561 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1621 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1681 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1741 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1801 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1861 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1921 ttctttcctg cgttatcccc tgattctgtg gataaccgtc gattatcaaa aaggatcttc
     1981 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2041 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2101 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2161 ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat
     2221 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2281 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2341 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2401 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2461 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2521 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     2581 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2641 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     2701 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     2761 ccgctgttga gatccagttc gatataaccc actcgtgcac ccaactgatc ttcagcatct
     2821 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     2881 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     2941 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3001 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3061 attattatca tgacattaac ctataaaaat aggcgtatca cgaggcagaa tttcagataa
     3121 aaaaaatcct tagctttcgc taaggatgat ttctggaatt cgcggccgct tctaga
//
