LOCUS       pWN212._pBBR1.wt_MSCA_# 3646 bp ds-DNA     circular     27-JUN-2023
DEFINITION  .
KEYWORDS    "marker:NeoR/KanR"
COMMENT     pBbB1k-GFP sequence 4727 bps
FEATURES             Location/Qualifiers
     misc_feature    1..6
                     /label="AatII"
                     /ApEinfo_revcolor="#ff00ff"
                     /ApEinfo_fwdcolor="#ff00ff"
                     /note="note: Restriction site used by the Bgl-brick team to define the BEGINNING of the inducible expression casette, which spans from the repressor up to the terminator of cds."
     misc_feature    7..67
                     /label="L3S2P21"
                     /ApEinfo_revcolor="#000000"
                     /ApEinfo_fwdcolor="#000000"
                     /note="note: Voigt's strongest tested synthetic promoter"
     misc_feature    7..250
                     /label="gBlocks part 1"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    68..87
                     /label="AT-rich random 20-bp"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    88..93
                     /label=-35
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     misc_feature    88..122
                     /label="gBlocks part 1"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    88..122
                     /label="J23107"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     misc_feature    111..116
                     /label=-10
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     misc_feature    123..123
                     /label="TSS"
                     /ApEinfo_revcolor="#ff0000"
                     /ApEinfo_fwdcolor="#ff0000"
     misc_feature    123..156
                     /label="HP4"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     misc_feature    174..179
                     /label="RBS"
                     /ApEinfo_revcolor="#ff0000"
                     /ApEinfo_fwdcolor="#ff0000"
                     /note="note: RBS"
     misc_feature    188..190
                     /label="START"
                     /ApEinfo_revcolor="#ff0000"
                     /ApEinfo_fwdcolor="#ff0000"
     misc_feature    194..223
                     /label="10 HisTag"
                     /ApEinfo_revcolor="#0080ff"
                     /ApEinfo_fwdcolor="#0080ff"
                     /note="note: His Tag"
     misc_feature    224..250
                     /label="Floppy linker"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: Linking Histag with the protein, very likely in a manner that minimizes any steric effect of the Histag to the protein, which can potentially affect the conformation thus functionality of the protein. Actually the length of this extended HisTag affects positively the overall insulation purpose of the tag."
     CDS             251..961
                     /label="sfGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="codon_start: 1 note: sfGFP transl_table: 1 translation: SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK"
     misc_feature    962..964
                     /label="STOP"
                     /ApEinfo_revcolor="#ff0000"
                     /ApEinfo_fwdcolor="#ff0000"
     misc_feature    965..973
                     /label="Spacer"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: Spacer as is found in the construct used by Voigt team to test their terminator library."
     terminator      974..1063
                     /label="ECK120029600"
                     /ApEinfo_revcolor="#000000"
                     /ApEinfo_fwdcolor="#000000"
                     /note="note: Voigt's strongest tested natural promoter"
     misc_feature    1064..1069
                     /label="AvrII"
                     /ApEinfo_revcolor="#ff00ff"
                     /ApEinfo_fwdcolor="#ff00ff"
     misc_feature    1064..2685
                     /label="Taylor's pBBR1.wt"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     CDS             complement(1124..1786)
                     /label="pBBR1 Rep"
                     /ApEinfo_revcolor="#993366"
                     /ApEinfo_fwdcolor="#993366"
                     /note="codon_start: 1 experiment: gene: note: product: replication protein for the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica protein_id: transl_table: 1 translation: MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLHHLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVVNDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEEPPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR*"
     rep_origin      1787..2557
                     /label="pBBR1 oriV"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="note: replication origin of the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica; requires the pBBR1 Rep protein for replication"
     misc_feature    2360..2579
                     /label="Missing from MS_const."
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     terminator      2591..2685
                     /label="lambda t0 terminator"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="gene: note: transcription terminator from phage lambda"
     misc_feature    2691..2696
                     /label="SacI"
                     /ApEinfo_revcolor="#ff00ff"
                     /ApEinfo_fwdcolor="#ff00ff"
     CDS             complement(2716..3510)
                     /label="NeoR/KanR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="codon_start: 1 gene: aph(3')-II (or nptII) note: confers resistance to neomycin, kanamycin, and G418 (Geneticin®) product: aminoglycoside phosphotransferase from Tn5 transl_table: 1 translation: MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
     promoter        complement(3599..3646)
                     /label="NEOKAN_promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
ORIGIN
        1 gacgtcCTCG GTACCAAATT CCAGAAAAGA GGCCTCCCGA AAGGGGGGCC TTTTTTCGTT
       61 TTGGTCCATC TTGAACTAGC ATACATAttt acggctagct cagccctagg tattatgcta
      121 gcacgtcgac ttatCTcGaG Tgagatattg ttgacgaact cataaagtca agtaggagat
      181 taattccATG ggccatcacc atcaccatca ccatcaccat cacagcagcg gccatatcga
      241 aggtcgtcat AGCAAAGGAG AAGAACTTTT CACTGGAGTT GTCCCAATTC TTGTTGAATT
      301 AGATGGTGAT GTTAATGGGC ACAAATTTTC TGTCCGTGGA GAGGGTGAAG GTGATGCTAC
      361 AAACGGAAAA CTCACCCTTA AATTTATTTG CACTACTGGA AAACTACCTG TTCCGTGGCC
      421 AACACTTGTC ACTACTCTGA CCTATGGTGT TCAATGCTTT TCCCGTTATC CGGATCACAT
      481 GAAACGGCAT GACTTTTTCA AGAGTGCCAT GCCCGAAGGT TATGTACAGG AACGCACTAT
      541 ATCTTTCAAA GATGACGGGA CCTACAAGAC GCGTGCTGAA GTCAAGTTTG AAGGTGATAC
      601 CCTTGTTAAT CGTATCGAGT TAAAGGGTAT TGATTTTAAA GAAGATGGAA ACATTCTTGG
      661 ACACAAACTT GAGTACAACT TTAACTCACA CAATGTATAC ATCACGGCAG ACAAACAAAA
      721 GAATGGAATC AAAGCTAACT TCAAAATTCG CCACAACGTT GAAGATGGTT CCGTTCAACT
      781 AGCAGACCAT TATCAACAAA ATACTCCAAT TGGCGATGGC CCTGTCCTTT TACCAGACAA
      841 CCATTACCTG TCGACACAAT CTGTCCTTTC GAAAGATCCC AACGAAAAGC GTGACCACAT
      901 GGTCCTTCTT GAGTTTGTAA CTGCTGCTGG GATTACACAT ggcatggatg agctctacaa
      961 ataataagaa ttcTTCAGCC AAAAAACTTA AGACCGCCGG TCTTGTCCAC TACCTTGCAG
     1021 TAATGCGGTG GACAGGATCG GCGGTTTTCT TTTCTCTTCT CAAcctaggc tacagccgat
     1081 agtctggaac agcgcactta cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg
     1141 tgacccgtgt cggcggctcc aacggctcgc catcgTCCAG AAAACACGGC TCATCgggca
     1201 tcggcaggcg ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt
     1261 ctggttccat gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta
     1321 gttgctgctc gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg
     1381 attcgtcctg gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact
     1441 ggtcgcgggg ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg
     1501 ggccgttgag cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt
     1561 ggaccgtccg caaagaacgt ccgatgagct tggaaagtgt cttctggctg accaccacgg
     1621 cgttctggtg gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc
     1681 tcgcaataag cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg
     1741 gcttgttctt ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc
     1801 ggtagggtgc cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg
     1861 acaacaatta tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca
     1921 atgagctatt gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt
     1981 gatttttaag tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca
     2041 cccctgcggg gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct
     2101 ccggggcttg ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt
     2161 ggaacccccg cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgccCttcg
     2221 actgccccca ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc
     2281 ggtcgctgcg cgagccttga cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg
     2341 caggccgcag gccggaggct tttccccaga gaaaattaaa aaaattgatg gggcaaggcc
     2401 gcaggccgcg cagttggagc cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa
     2461 tccctgtggt caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc agcagggttg
     2521 tccacgggcc gagcgaagcg agccagccgg tggccgctcG CGGCCATCGT CCACATATCc
     2581 tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
     2641 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctctcga
     2701 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     2761 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     2821 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     2881 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     2941 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
     3001 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     3061 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     3121 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     3181 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     3241 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     3301 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     3361 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     3421 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     3481 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
     3541 agatcatgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
     3601 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattcc
//