LOCUS       sequence-265023-        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"
     rep_origin      263..698
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             complement(735..1706)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSYFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     primer_bind     2510..2527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2551..2608)
                     /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."
     misc_feature    2609..2629
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      complement(2648..2675)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2691..2762)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(2779..3492,3493..3495))
                     /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             complement(2782..3495)
                     /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(2822..2842)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3437..3465
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     misc_RNA        complement(3538..3587)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(3595..3629)
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     misc_feature    3650..3671
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      3674..3717
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(3721..3739)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3807..3911
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3912..3980,3981..4772)
                     /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     complement(4130..4149)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgtttagtga ttgccattct tgcgggcctc tgtactgtag tatgttgtat gatactacat
       61 actacaacaa tttaacagag ccatcttgga atctggtgtc tctgcgccta taattctgga
      121 acagctactt tccgaacgac tcctgcgttg atcggaaatc cagaagcccg agaggttgcc
      181 gcctttcggg ctttttcttt ttcaaaaaaa aaaatttata aaacgatctg ttgcggccgc
      241 cgggttgtgg gcaaaggcgc tggcgctcga cggtgggcaa ccgcttgcgg ttgtccacgg
      301 gcggagccgg tgcgcgtagc gcattgtcca caagccaagg gcgaccaata attgatatat
      361 atattcataa ttgaaaagct aattgaacat actacttgct gtaactactt gccggagcga
      421 ggggtgtttg caagctgttg atctgaaagg gctattagcg ttctcacgtg cctttttgat
      481 tagcgatttc acgtgacctt attagcgatt tcacgtactc cgattagcga tttcacgtac
      541 cctgattagc gatttcacgt ggatagtttt tggagcgggc cggaaagccc cgtgaatcaa
      601 ggctttgcgg ggcattagcg gtttcacgtg gataactacc ctctatccac aggcttccgg
      661 ggataaaaaa gcccgctcga cggcgggctg ttggatggga aggcttgacc aagccaagcg
      721 tagcgttggc ctggtcaagt cggagggggg ccgatgcgag cgcccttgcc gggtgcgcgg
      781 gtgacatgca ggcgtgtgga tttgatgcgc aggcattcgc cgtcatcttc gatgcagtcg
      841 cttgcctcgg gatagacaat caacacttcg cgtaggcgct ttttgaagtt gtatttgaag
      901 ctggcgagtg ctgcccgctc tgcccgctct cgggccttat cgtccagttc gggcgagttg
      961 cgtgcgcggc tgccatagga tgagccgaat tgcgcttgca gggcgaccca agggatttgc
     1021 acgaaggggc ggcccttggc ccgcaacagg aacacgcgat aggtcagcca cgtgtaaatg
     1081 tccatcgcaa gcggagactg ccgcaaggca tgcaggtagt cgattcggat aggaaccggt
     1141 gagcgggtga cttcctcgaa gaaatcgcct gtgagggtga gggtgctatc ccatagcgcc
     1201 cgatcttctg gccgcttggg attccagaat agaaaagcgc gcttggcaat gacgacgttc
     1261 tcaatgccga agtcattgcc ttgctcgccg gcaagcgaaa tcatggatga aaacaggcgt
     1321 tgcgcctgat tgcgaagggt ggccgtgtaa cggccatcgg tgtgcattcc gagcctttgt
     1381 agaaattccg attgcgaccg gccaaggttc aacacggggt ctttcgttcg cacggcctcg
     1441 gtgcatatcc aagcaagcaa ggtgcgcggc atagaaccgt agggcaggcc gatgctcggc
     1501 ttgcccatga tcgacaaggt gacgatgcca ttggtgcgct caaagtagct ggtcttgggg
     1561 tcggtgtggg gcatggtcgc ttgcacaagg caacgggcca tgtagccgac taagccagct
     1621 tcgcgggcat cctccatttc gagcgcgagg ctcgtcttga tgatctcgtt gatacgatgg
     1681 ccgggggctt tgttgttctt aggcatgttg ttccctcccc ggcatggtga tggttggtct
     1741 agtgtttgtg ggtttgatgt tccggcgttt gatgaacagg cgcaaggtgt gagggctgac
     1801 gcctaacaac tcggctgcgc gactttgcgg caagccaagg ttcacgtatg cctgtacttc
     1861 atcaatacgg ctgtccagct tcaaggcgct cgatttgctg cccttgggtc gcccgagcgt
     1921 cttgccgcgc tctctggcga cttgtagcgc ctcggtggta cgtgcctgaa tgaaatgccg
     1981 ctcgatctgt gcagccaagc caagcacggt tgccatgatg tcgctttgta ggctgccgtc
     2041 catgatgatc ttctgtttgg tcacatggac gattaggccg cgctcgctcg ccgctttgag
     2101 aatttccaag gcggcgaggg cggaaccggc aatgcgcgta atctccggcg tcagtagcac
     2161 gtcgccacgc tcggcctttt cgatgattgc tccgagcttg cgcttgcgcc agtcctttgc
     2221 tctgctggca atttcttcct cgatctgtag cggcgcgaag cctttggcgt tcgcgtattc
     2281 gagcaaaccg tatttttggt tttccgggtc ttggccgtca cgcgaaaccc ggagataggc
     2341 atagtatttt ggcatttgca gggaaaacgt cagattcggt taaacatgcc tcattctagc
     2401 gcagattaaa taggaattaa ataccctgta gcggtataga taaaacgttg gtttgattac
     2461 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2521 gagcgaggaa gcctgcataa cgcgaagtaa tcttttcggt tttaaagaaa aagggcaggg
     2581 tggtgacacc ttgccctttt ttgccggact gcagcggccg ctactagtaa gcgatgtctt
     2641 ctataaacgc agaaaggccc acccgaaggt gagccagtgt gactctagta gagagcgttc
     2701 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     2761 tgatgcctgg ctctagtatt atttgtagag ctcatccatg ccatgtgtaa tcccagcagc
     2821 agttacaaac tcaagaagga ccatgtggtc acgcttttcg ttgggatctt tcgaaaggac
     2881 agattgtgtc gacaggtaat ggttgtctgg taaaaggaca gggccatcgc caattggagt
     2941 attttgttga taatggtctg ctagttgaac ggaaccatct tcaacgttgt ggcgaatttt
     3001 gaagttagct ttgattccat tcttttgttt gtctgccgtg atgtatacat tgtgtgagtt
     3061 aaagttgtac tcgagtttgt gtccaagaat gtttccatct tctttaaaat caataccctt
     3121 taactcgata cgattaacaa gggtatcacc ttcaaacttg acttcagcac gcgtcttgta
     3181 ggtcccgtca tctttgaaag atatagtgcg ttcctgtaca taaccttcgg gcatggcact
     3241 cttgaaaaag tcatgccgtt tcatgtgatc cggataacgg gaaaagcatt gaacaccata
     3301 ggtcagagta gtgacaagtg ttggccacgg aacaggtagt tttccagtag tgcaaataaa
     3361 tttaagggtg agttttccgt ttgtagcatc accttcaccc tctccacgga cagaaaattt
     3421 gtgcccatta acatcaccat ctaattcaac aagaattggg acaactccag tgaaaagttc
     3481 ttctcctttg ctcatctagt actttcctgt gtgattaaac aaaattattt gtagaggctg
     3541 tttcgtcctc acggactcat cagaccggaa agcacatccg gtgacagcta ccttgctagc
     3601 atagtaccta ggactgagct agccgtaaag gcgcgccgaa gacacctccc tctagaagcg
     3661 gccgcgaatt ccagaaatca tccttagcga aagctaagga ttttttttat ctgaaattct
     3721 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3781 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3841 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3901 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3961 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4021 agttgggtgc acgagtgggt tatatcgaac tggatctcaa cagcggtaag atccttgaga
     4081 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4141 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4201 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4261 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4321 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4381 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4441 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4501 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4561 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4621 agcgtggttc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4681 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4741 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4801 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4861 ataatcggcg ctgtgcgcta tggcgatctg gaagtgtcgg gacgaggggc gtcccggcgg
     4921 taaattcgac gtgcctgcgg cgcgtcgaac aaggggcgtg cccggtgtca gttcgtggca
     4981 tttttcgagg cgcgacgcca tttccaaggc tcctgagcat tcgggtctga ccaaaggccg
     5041 agccgttggc ggcgggcctc tttttcatac tcgtacatct gcgcgtcggt tggtttgccg
     5101 taataacggt aagcccaggc catgccttct tgaaccatga tcgcattgat gttggtgagt
     5161 tgtgtttggc cgccggggta ttgcaacggc gcgtaaacga ccccaagagt gcggccatac
     5221 cgatcaacct ctttttcggt cacttgaacc tcttggcgaa aggtcaagtc ggcgagccgt
     5281 tggcgagcac gggagccgaa ggcttggccg ctttccggtg cgtcaatatc ggccaatctc
     5341 acgcggatgg tctgacggtt caccaaaacg tcgatagtgt caccgtcaag gattcggacg
     5401 acttcacccc ggaagtcggc ccaagcgggc acactgacga ttaggacgac agcggccgcg
     5461 accgcgcgaa gggcggcaag ggcgcttttc attgtttgcc tcctgttttc aagacggctg
     5521 tgagattggc gacctgctct ttgagggctt ccacctgacc ttgcagactg gcggcgcgct
     5581 cgatggcctc cttggcctgt ttgcgggcct cgatagcctc gttatcgcgt tgggtgagct
     5641 tttccatgca gcggtttagt tcttcgccgc tgcggcgttt cacttcggca agctggtcgg
     5701 ccagcttgtc gcgctcgcgt tccataggtt cgagctgatt cactcgttcg cggagctggt
     5761 cgttttcgcg ggtgaaggtg tcggctagtt cgattgcttc ggcaagctgc tggctgatgg
     5821 ccgctttgtc ggcctcgatc tgtttccgat cttcgtcaaa ccgggcgttg gcgtgcgcca
     5881 gggcgatagc ccatagcgca ttgccaagct cggcaagatg ctcgttgact gcaaccggca
     5941 atgggtctga tgagggcagg gtggcggtct tgcggttttt ccattcagcc attgcatcgg
     6001 aaatggttgt gaagctaccg cttccgagtt tcttgcgcac ggcggccaaa gtgggccgga
     6061 tgccttcggc gtccagttcg tcggctgctc gccaaatgtc t
//
