LOCUS       sequence-206001-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  incW BbsI cloning vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  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 6101)
  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_50
FEATURES             Location/Qualifiers
     source          1..6101
                     /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"
     CDS             1944..2915
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSYFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     rep_origin      2952..3387
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             complement(join(4979..5770,5771..5839))
                     /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     5602..5621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5840..5944)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6012..6030
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(6034..6077)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    6080..6101
                     /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 taatcaaacc
     1201 aacgttttat ctataccgct acagggtatt taattcctat ttaatctgcg ctagaatgag
     1261 gcatgtttaa ccgaatctga cgttttccct gcaaatgcca aaatactatg cctatctccg
     1321 ggtttcgcgt gacggccaag acccggaaaa ccaaaaatac ggtttgctcg aatacgcgaa
     1381 cgccaaaggc ttcgcgccgc tacagatcga ggaagaaatt gccagcagag caaaggactg
     1441 gcgcaagcgc aagctcggag caatcatcga aaaggccgag cgtggcgacg tgctactgac
     1501 gccggagatt acgcgcattg ccggttccgc cctcgccgcc ttggaaattc tcaaagcggc
     1561 gagcgagcgc ggcctaatcg tccatgtgac caaacagaag atcatcatgg acggcagcct
     1621 acaaagcgac atcatggcaa ccgtgcttgg cttggctgca cagatcgagc ggcatttcat
     1681 tcaggcacgt accaccgagg cgctacaagt cgccagagag cgcggcaaga cgctcgggcg
     1741 acccaagggc agcaaatcga gcgccttgaa gctggacagc cgtattgatg aagtacaggc
     1801 atacgtgaac cttggcttgc cgcaaagtcg cgcagccgag ttgttaggcg tcagccctca
     1861 caccttgcgc ctgttcatca aacgccggaa catcaaaccc acaaacacta gaccaaccat
     1921 caccatgccg gggagggaac aacatgccta agaacaacaa agcccccggc catcgtatca
     1981 acgagatcat caagacgagc ctcgcgctcg aaatggagga tgcccgcgaa gctggcttag
     2041 tcggctacat ggcccgttgc cttgtgcaag cgaccatgcc ccacaccgac cccaagacca
     2101 gctactttga gcgcaccaat ggcatcgtca ccttgtcgat catgggcaag ccgagcatcg
     2161 gcctgcccta cggttctatg ccgcgcacct tgcttgcttg gatatgcacc gaggccgtgc
     2221 gaacgaaaga ccccgtgttg aaccttggcc ggtcgcaatc ggaatttcta caaaggctcg
     2281 gaatgcacac cgatggccgt tacacggcca cccttcgcaa tcaggcgcaa cgcctgtttt
     2341 catccatgat ttcgcttgcc ggcgagcaag gcaatgactt cggcattgag aacgtcgtca
     2401 ttgccaagcg cgcttttcta ttctggaatc ccaagcggcc agaagatcgg gcgctatggg
     2461 atagcaccct caccctcaca ggcgatttct tcgaggaagt cacccgctca ccggttccta
     2521 tccgaatcga ctacctgcat gccttgcggc agtctccgct tgcgatggac atttacacgt
     2581 ggctgaccta tcgcgtgttc ctgttgcggg ccaagggccg ccccttcgtg caaatccctt
     2641 gggtcgccct gcaagcgcaa ttcggctcat cctatggcag ccgcgcacgc aactcgcccg
     2701 aactggacga taaggcccga gagcgggcag agcgggcagc actcgccagc ttcaaataca
     2761 acttcaaaaa gcgcctacgc gaagtgttga ttgtctatcc cgaggcaagc gactgcatcg
     2821 aagatgacgg cgaatgcctg cgcatcaaat ccacacgcct gcatgtcacc cgcgcacccg
     2881 gcaagggcgc tcgcatcggc ccccctccga cttgaccagg ccaacgctac gcttggcttg
     2941 gtcaagcctt cccatccaac agcccgccgt cgagcgggct tttttatccc cggaagcctg
     3001 tggatagagg gtagttatcc acgtgaaacc gctaatgccc cgcaaagcct tgattcacgg
     3061 ggctttccgg cccgctccaa aaactatcca cgtgaaatcg ctaatcaggg tacgtgaaat
     3121 cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg aaatcgctaa tcaaaaaggc
     3181 acgtgagaac gctaatagcc ctttcagatc aacagcttgc aaacacccct cgctccggca
     3241 agtagttaca gcaagtagta tgttcaatta gcttttcaat tatgaatata tatatcaatt
     3301 attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac cggctccgcc cgtggacaac
     3361 cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc cacaacccgg cggccgcaac
     3421 agatcgtttt ataaattttt ttttttgaaa aagaaaaagc ccgaaaggcg gcaacctctc
     3481 gggcttctgg atttccgatc aacgcaggag tcgttcggaa agtagctgtt ccagaattat
     3541 aggcgcagag acaccagatt ccaagatggc tctgttaaat tgttgtagta tgtagtatca
     3601 tacaacatac tacagtacag aggcccgcaa gaatggcaat cactaaacaa gacatttggc
     3661 gagcagccga cgaactggac gccgaaggca tccggcccac tttggccgcc gtgcgcaaga
     3721 aactcggaag cggtagcttc acaaccattt ccgatgcaat ggctgaatgg aaaaaccgca
     3781 agaccgccac cctgccctca tcagacccat tgccggttgc agtcaacgag catcttgccg
     3841 agcttggcaa tgcgctatgg gctatcgccc tggcgcacgc caacgcccgg tttgacgaag
     3901 atcggaaaca gatcgaggcc gacaaagcgg ccatcagcca gcagcttgcc gaagcaatcg
     3961 aactagccga caccttcacc cgcgaaaacg accagctccg cgaacgagtg aatcagctcg
     4021 aacctatgga acgcgagcgc gacaagctgg ccgaccagct tgccgaagtg aaacgccgca
     4081 gcggcgaaga actaaaccgc tgcatggaaa agctcaccca acgcgataac gaggctatcg
     4141 aggcccgcaa acaggccaag gaggccatcg agcgcgccgc cagtctgcaa ggtcaggtgg
     4201 aagccctcaa agagcaggtc gccaatctca cagccgtctt gaaaacagga ggcaaacaat
     4261 gaaaagcgcc cttgccgccc ttcgcgcggt cgcggccgct gtcgtcctaa tcgtcagtgt
     4321 gcccgcttgg gccgacttcc ggggtgaagt cgtccgaatc cttgacggtg acactatcga
     4381 cgttttggtg aaccgtcaga ccatccgcgt gagattggcc gatattgacg caccggaaag
     4441 cggccaagcc ttcggctccc gtgctcgcca acggctcgcc gacttgacct ttcgccaaga
     4501 ggttcaagtg accgaaaaag aggttgatcg gtatggccgc actcttgggg tcgtttacgc
     4561 gccgttgcaa taccccggcg gccaaacaca actcaccaac atcaatgcga tcatggttca
     4621 agaaggcatg gcctgggctt accgttatta cggcaaacca accgacgcgc agatgtacga
     4681 gtatgaaaaa gaggcccgcc gccaacggct cggcctttgg tcagacccga atgctcagga
     4741 gccttggaaa tggcgtcgcg cctcgaaaaa tgccacgaac tgacaccggg cacgcccctt
     4801 gttcgacgcg ccgcaggcac gtcgaattta ccgccgggac gcccctcgtc ccgacacttc
     4861 cagatcgcca tagcgcacag cgccgattat caaaaaggat cttcacctag atccttttaa
     4921 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4981 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5041 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5101 gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca gcaataaacc
     5161 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5221 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5281 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5341 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5401 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5461 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5521 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5581 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5641 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5701 gttcgatata acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5761 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5821 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5881 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5941 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6001 taacctataa aaataggcgt atcacgaggc agaatttcag ataaaaaaaa tccttagctt
     6061 tcgctaagga tgatttctgg aattcgcggc cgcttctaga g
//
