LOCUS       sequence-128125-        4359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing CCSP for generation of GMAP-compatible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4359)
  AUTHORS   Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL, Lee DY, 
            Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T
  TITLE     A Modular Assembly Platform for Rapid Generation of DNA Constructs.
  JOURNAL   Sci Rep. 2016 Feb 18;6:16836. doi: 10.1038/srep16836.
  PUBMED    26887506
REFERENCE   2  (bases 1 to 4359)
  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: "Sci Rep."; 
            date: "2016-02-18"; volume: "6:16836. doi"; pages: " 
            10.1038/srep16836"
COMMENT     Sequence Label: TOPO CCSP
FEATURES             Location/Qualifiers
     source          1..4359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1246..1265)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1247..1265)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1272..1289)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1272..1288)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1281..1303)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1426..1728
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1607..1626
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2077..2871
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2077..2871
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2131..2150)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2741..2760
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3072..3081
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3078..3452
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3590..4178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4079..4098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4332..4349
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcag
      301 tgtgagggag tgtaaagctg gtttcctggg aatggctaac acttgagaac tgtcaacatc
      361 atgaaaggat aagaaagaat ggccaaggaa agaaaacagg aaagagctga gtgtgggaga
      421 ggcctggaga gaatggggca agaagtggga ggctaaaggg taagggcaag ggagggtcaa
      481 gtcagatgag gactgaaggt gctttcaact ggtaggactt gagggctgct ctgggtagga
      541 gccagcccgg tctgggccct gggtctctca tgtgttctat ggagaagtct ttatatttgt
      601 tcatgtgtat tgtatgtggg ctctaacctg gccaacaatg cccaagaatc aagtgatttt
      661 tgtggcttga agtctagcca ctttccttgg agggaggcaa tagaaggagc ctagtgacat
      721 ctcggacgga gtctggggtc tttgtccttc cccttgattc ctgaagggtc tccagcctct
      781 ggttctccag ggttggcaag tctacaattg cttcccggaa cctggagtgc tcagtgcttg
      841 actttaaaga ggacacaggt gcctacagtt ccacgacctc tgggttcccc gacgcccccc
      901 cccccgcact gcccattgcc caaacacccc acaagtggcc tattgtgtga gctcagtttc
      961 aatgggaaaa gaaactgggt ttatgaaaag agattatttg cttattgcat ggagatgact
     1021 aagtaaatag tgcaatttct tgagtggagc acaatccctg cccctacctc ttgtgggctg
     1081 ccaggaacat ataaaaagcc acacgcatac ccacacatac ccgacccgac attagcgcta
     1141 cagcttaagc gggatccact agtaacggcc gccagtgtgc tggaattctg cagatatcca
     1201 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
     1261 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1321 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1381 cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac
     1441 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1501 acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1561 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1621 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     1681 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg
     1741 agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa
     1801 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     1861 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     1921 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     1981 ccctgcaaag taaactggat ggctttctcg ccgccaagga tctgatggcg caggggatca
     2041 agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2101 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2161 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2221 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2281 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2341 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2401 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2461 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2521 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2581 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     2641 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2701 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2761 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2821 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac
     2881 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2941 atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3001 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc
     3061 acgtgaggag ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     3121 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     3181 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     3241 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     3301 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     3361 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     3421 gcgtgcactt cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta
     3481 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3541 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3601 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3661 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3721 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3781 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3841 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3901 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3961 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4021 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4081 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4141 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4201 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4261 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4321 cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
