LOCUS       sequence-128085-        4513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing CloverCP for generation of GMAP-compatible
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4513)
  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 4513)
  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 CloverCP
FEATURES             Location/Qualifiers
     source          1..4513
                     /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"
     regulatory      337..346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(343..345,346..348,349..1059)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(388..409)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     996..1017
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1075..1093)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             1102..1149
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             1153..1272
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     primer_bind     complement(1400..1419)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1401..1419)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1426..1443)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1426..1442)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1435..1457)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1580..1882
                     /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     1761..1780
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2231..3025
                     /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             2231..3025
                     /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(2285..2304)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2895..2914
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3226..3235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3232..3606
                     /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      3744..4332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4233..4252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4486..4503
                     /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 ctcggatcct
      301 aactcgaacg ctagctgtgc gatcgttttt aattaagcca ccatggtgag caagggcgag
      361 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      421 aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
      481 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
      541 tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
      601 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga cgacggtacc
      661 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      721 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc
      781 aacagccaca acgtctatat cacggccgac aagcagaaga acggcatcaa ggctaacttc
      841 aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
      901 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ccatcagtcc
      961 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1021 gccgccggga ttacacatgg catggacgag ctgtacaagt ctagagggcc ctattctata
     1081 gtgtcaccta aatgcaattc tgcttgcaag aactggttca gtagcttaag ccactttgtg
     1141 atccacctta acagccacgg cttccctccc gaggtggagg agcaggccgc cggcaccctg
     1201 cccatgagct gcgcccagga gagcggcatg gatagacacc ctgctgcttg cgccagcgcc
     1261 aggatcaacg tctaaaaacg ttgttgtttg gggttgaatt actctggatc cactagtaac
     1321 ggccgccagt gtgctggaat tctgcagata tccatcacac tggcggccgc tcgagcatgc
     1381 atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     1441 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1501 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1561 gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     1621 tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc
     1681 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     1741 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     1801 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     1861 cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta
     1921 gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
     1981 ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag
     2041 tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt
     2101 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
     2161 ctcgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg
     2221 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2281 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2341 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2401 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2461 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2521 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     2581 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     2641 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     2701 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     2761 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     2821 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     2881 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     2941 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3001 tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt
     3061 tctccttacg catctgtgcg gtatttcaca ccgcatacag gtggcacttt tcggggaaat
     3121 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3181 agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag
     3241 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     3301 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     3361 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3421 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3481 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3541 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3601 gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3661 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3721 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3781 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3841 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3901 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3961 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4021 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4081 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4141 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4201 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4261 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4321 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4381 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4441 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4501 gaggaagcgg aag
//
