LOCUS       sequence-206003-        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        21..55
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     misc_RNA        63..112
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(155..157,158..871)
                     /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             155..868
                     /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(185..213)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     808..828
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      888..959
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      975..1002
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1021..1041
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1042..1099
                     /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(1123..1140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1271..1859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1760..1779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2054..2845,2846..2914))
                     /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     2677..2696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2915..3019)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3087..3105
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3109..3152)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3155..3176
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ggaggtgtct tcggcgcgcc tttacggcta gctcagtcct aggtactatg ctagcaaggt
       61 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
      121 aataattttg tttaatcaca caggaaagta ctagatgagc aaaggagaag aacttttcac
      181 tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt
      241 ccgtggagag ggtgaaggtg atgctacaaa cggaaaactc acccttaaat ttatttgcac
      301 tactggaaaa ctacctgttc cgtggccaac acttgtcact actctgacct atggtgttca
      361 atgcttttcc cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc
      421 cgaaggttat gtacaggaac gcactatatc tttcaaagat gacgggacct acaagacgcg
      481 tgctgaagtc aagtttgaag gtgataccct tgttaatcgt atcgagttaa agggtattga
      541 ttttaaagaa gatggaaaca ttcttggaca caaactcgag tacaacttta actcacacaa
      601 tgtatacatc acggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattcgcca
      661 caacgttgaa gatggttccg ttcaactagc agaccattat caacaaaata ctccaattgg
      721 cgatggccct gtccttttac cagacaacca ttacctgtcg acacaatctg tcctttcgaa
      781 agatcccaac gaaaagcgtg accacatggt ccttcttgag tttgtaactg ctgctgggat
      841 tacacatggc atggatgagc tctacaaata atactagagc caggcatcaa ataaaacgaa
      901 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct
      961 actagagtca cactggctca ccttcgggtg ggcctttctg cgtttataga agacatcgct
     1021 tactagtagc ggccgctgca gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt
     1081 ttctttaaaa ccgaaaagat tacttcgcgt tatgcaggct tcctcgctca ctgactcgct
     1141 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taattcatga
     1201 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1261 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1321 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1381 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     1441 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1501 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1561 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1621 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1681 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1741 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1801 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1861 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1921 tctttcctgc gttatcccct gattctgtgg ataaccgtcg attatcaaaa aggatcttca
     1981 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2041 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2101 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2161 taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt
     2221 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2281 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2341 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2401 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2461 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2521 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2581 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2641 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2701 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2761 cgctgttgag atccagttcg atataaccca ctcgtgcacc caactgatct tcagcatctt
     2821 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2881 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2941 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3001 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     3061 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcagaat ttcagataaa
     3121 aaaaatcctt agctttcgct aaggatgatt tctggaattc gcggccgctt ctagag
//
