LOCUS       sequence-414115-        3674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p15A-tetR-tet-SapI-ME-RBS-mRFP-ME-U1-Barcode-U2-SapI-dbI 
            terminator-oriT-Cm. Fragment expression vector with Golden Gate 
            sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3674)
  AUTHORS   Biggs BW, Price MN, Lai D, Escobedo J, Fortanel Y, Huang YY, Kim K, 
            Trotter VV, Kuehl JV, Lui LM, Chakraborty R, Deutschbauer AM, Arkin 
            AP
  TITLE     High-throughput protein characterization by complementation using 
            DNA barcoded fragment libraries.
  JOURNAL   Mol Syst Biol. 2024 Oct 7. doi: 10.1038/s44320-024-00068-z.
  PUBMED    39375541
REFERENCE   2  (bases 1 to 3674)
  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: "Mol Syst 
            Biol. 2024 Oct 7. doi: 10.1038/s44320-024-00068-z."
COMMENT     Sequence Label: pBWB514
FEATURES             Location/Qualifiers
     source          1..3674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..627)
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     532..551
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        643..698
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    679..697
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    714..732
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             753..1430
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     misc_feature    1431..1449
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      1569..1640
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1656..1683
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1709..1726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1843..2388)
                     /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(1923..1942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            2473..2581
                     /note="incP origin of transfer"
     terminator      complement(2768..2862)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2883..3542)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3457..3476
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3543..3645)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttaagaccca ctttcacatt taagttgttt ttctaatccg catatgatca attcaaggcc
       61 gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa
      121 tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg
      181 atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt
      241 ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg
      301 tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc
      361 acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc cttctaaagg
      421 gcaaaagtga gtatggtgcc tatctaacat ctcaatggct aaggcgtcga gcaaagcccg
      481 cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct gggcgagttt
      541 acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac
      601 tttactttta tctaatctag acatcattaa ttcctaattt ttgttgacac tctatcgttg
      661 atagagttat tttaccactc cctatcagtg atagagaaaa gctcttcagt ccaagatgtg
      721 tataagagac agtttaagaa ggagatatac atatggcgag tagcgaagac gttatcaaag
      781 agttcatgcg tttcaaagtt cgtatggaag gttccgttaa cggtcacgag ttcgaaatcg
      841 aaggtgaagg tgaaggtcgt ccgtacgaag gtacccagac cgctaaactg aaagttacca
      901 aaggtggtcc gctgccgttc gcttgggaca tcctgtcccc gcagttccag tacggttcca
      961 aagcttacgt taaacacccg gctgacatcc cggactacct gaaactgtcc ttcccggaag
     1021 gtttcaaatg ggaacgtgtt atgaacttcg aagacggtgg tgttgttacc gttacccagg
     1081 actcctccct gcaagacggt gagttcatct acaaagttaa actgcgtggt accaacttcc
     1141 cgtccgacgg tccggttatg cagaaaaaaa ccatgggttg ggaagcttcc accgaacgta
     1201 tgtacccgga agacggtgct ctgaaaggtg aaatcaaaat gcgtctgaaa ctgaaagacg
     1261 gtggtcacta cgacgctgaa gttaaaacca cctacatggc taaaaaaccg gttcagctgc
     1321 cgggtgctta caaaaccgac atcaaactgg acatcacctc ccacaacgaa gactacacca
     1381 tcgttgaaca gtacgaacgt gctgaaggtc gtcactccac cggtgcttaa ctgtctctta
     1441 tacacatctg tcgacctgca gcgtacgtca ctaatggcca aataacaaga gacctcgtgg
     1501 acatcgtcag cgaagagcta gggataacag ggtaatggat ccaaactcga gtaaggatct
     1561 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1621 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     1681 gcgtttatac cgggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     1741 gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga
     1801 agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     1861 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     1921 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     1981 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     2041 tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt
     2101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa
     2161 aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc
     2221 gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta
     2281 cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt
     2341 ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt
     2401 attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacct
     2461 gttcttgctt atcggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga
     2521 ataagggaca gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc
     2581 cggctgacgc cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt
     2641 atatcgtgcg aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg
     2701 ttatgcagcg gaaaagaatg tcatgataag tcagccccat acgatataag ttgttactag
     2761 tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca
     2821 gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgatatca
     2881 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     2941 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     3001 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     3061 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgaaac gaaaaacata
     3121 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     3181 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     3241 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     3301 tcaccgtctt tcattgccat acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg
     3361 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     3421 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     3481 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     3541 attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     3601 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaaggtc tcattttcgc
     3661 cagatatcga cgtc
//
