LOCUS       sequence-254272-        6083 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses amcyan fluorescent gene and sRNA nt 539-559.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  TITLE     A novel synthetic sRNA promoting protein overexpression in cell-free
            systems
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6083)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pART15-asB539
FEATURES             Location/Qualifiers
     source          1..6083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             318..1007
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKMTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHAVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPF"
     primer_bind     complement(1091..1108)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1261..1347
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1439..1466
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    1586..1604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(join(1611..1636,1637..1642,1643..1659,1660..1665,
                     1666..1684))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1641..1659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1666..1684
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1820..1838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1845..1863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2255..2878
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2328..2347)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3136..3153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3270..3815)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3350..3369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3980..4771,4772..4840))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4603..4622
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4841..4932)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4980..4999
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5179..6057)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatactcg tttttttggg
      301 ctagcaggag gaaagctatg gctctttcaa acaagtttat cggagatgac atgaaaatga
      361 cctaccatat ggatggctgt gtcaatgggc attactttac cgtcaaaggt gaaggcagcg
      421 ggaagccata cgaagggacg cagacctcga cttttaaagt caccatggcc aacggtgggc
      481 cccttgcatt ctcctttgac atactatcta cagtgttcat gtatggaaat cgatgcttta
      541 ctgcgtatcc taccagtatg cccgactatt tcaaacaagc atttcctgac ggaatgtcat
      601 atgaaaggac ttttacctat gaagatggag gagttgctac agccagttgg gaaataagcc
      661 ttaaaggcaa ctgctttgag cacaaatcca cgtttcatgg agtgaacttt cctgctgatg
      721 gacctgtgat ggcgaagatg acaactggtt gggacccatc ttttgagaaa atgactgtct
      781 gcgatggaat attgaagggt gatgtcaccg cgttcctcat gctgcaagga ggtggcaatt
      841 acagatgcca attccacact tcttacaaga caaaaaaacc ggtgacgatg ccaccaaacc
      901 atgcggtgga acatcgcatt gcgaggaccg accttgacaa aggtggcaac agtgttcagc
      961 ttacggagca cgctgttgca catataacct ctgttgtccc tttctgacac catggtaccc
     1021 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg ctgttttggc ggatgagaga
     1081 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
     1141 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
     1201 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat
     1261 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     1321 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
     1381 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag
     1441 aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tgggcgcctg
     1501 tcactttgct tgataaagga ggtggcaatt acagatatga gaattattta accttactcg
     1561 agatctcctg ctgatgtgct cagtatctct atcactgata gggacagctg ggtcagtgcg
     1621 tcctgctgat gtgctcagta tctctatcac tgatagggat gtcaatctct atcactgata
     1681 gggacagctg ggatcctaaa tgagaattat ttaaccttac tcgagatctg atcttcagca
     1741 tcttttactt tcaccagccc tgctgatgtg ctcagaatta tttaacctta ctcgagatct
     1801 cctgctgatg tgctcagtat ctctatcact gatagggatg tcaatctcta tcactgatag
     1861 ggacagctgg gatcctaaat gagaaaaaag caacgcactt taaataagat acgttgcttt
     1921 ttcgattgat gaacacctat aattaaacta ttcatctatt atttatgatt ttttgtatat
     1981 acaatatttc tagtttgtta aagagaatta agaaaataaa tctcgaaaat aataaaggga
     2041 aaatcagttt ttgatatcaa aattatacat gtcaacgata atacaaaata taatacaaac
     2101 tataagatgt tatcagtatt tattatgcat ttagaataaa ttttgtgtcg ccctttacac
     2161 gtacttagtc gctgaacagc tgatcaatgt ggacttttct gccggcatgc cagttgtaga
     2221 tcagcatagc atagacaacc aaggaggaac agctatgtct cgtttagata aaagtaaagt
     2281 gattaacagc gcattagagc tgcttaatga ggtcggaatc gaaggtttaa caacccgtaa
     2341 actcgcccag aagctaggtg tagagcagcc tacattgtat tggcatgtaa aaaataagcg
     2401 ggctttgctc gacgccttag ccattgagat gttagatagg caccatactc acttttgccc
     2461 tttagaaggg gaaagctggc aagatttttt acgtaataac gctaaaagtt ttagatgtgc
     2521 tttactaagt catcgcgatg gagcaaaagt acatttaggt acacggccta cagaaaaaca
     2581 gtatgaaact ctcgaaaatc aattagcctt tttatgccaa caaggttttt cactagagaa
     2641 tgcattatat gcactcagcg ctgtggggca ttttacttta ggttgcgtat tggaagatca
     2701 agagcatcaa gtcgctaaag aagaaaggga aacacctact actgatagta tgccgccatt
     2761 attacgacaa gctatcgaat tatttgatca ccaaggtgca gagccagcct tcttattcgg
     2821 ccttgaattg atcatctgcg gattagaaaa acaacttaaa tgtgaaagtg ggtcttaaca
     2881 gctgcgtcaa aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact
     2941 gagcctttcg ttttatttga tgcctgggag tcagcaacac cttcttcacg aggcagacct
     3001 cagcgctagc ggagtgtata ctggcttact atgttggcac tgatgagggt gtcagtgaag
     3061 tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt cagcagaata tgtgatacag
     3121 gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc
     3181 ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag
     3241 ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat
     3301 cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag
     3361 gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt
     3421 gtcattccgc tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta
     3481 ggcagttcgc tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc
     3541 cttatccggt aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc
     3601 agcagccact ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct
     3661 aaactgaaag gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag
     3721 agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag
     3781 agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aaggggtctg
     3841 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3901 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3961 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4021 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4081 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4141 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4201 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4261 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     4321 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4381 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4441 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4501 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4561 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     4621 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4681 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4741 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4801 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4861 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4921 aaaataaaca aataggggtt gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4981 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5041 atgcgtccgg cgtagaggat ctgctcatgt ttgacagctt atcatcgatg cataatgtgc
     5101 ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat
     5161 tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca
     5221 accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata
     5281 gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct
     5341 caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc
     5401 taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac
     5461 gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg
     5521 tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa
     5581 caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc
     5641 gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa
     5701 gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg
     5761 acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg
     5821 aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg
     5881 atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag
     5941 cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac
     6001 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
     6061 tttcatactc ccgccattca gag
//
