LOCUS       sequence-128089-        5438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing rtTA3-2A-Cre for generation of 
            GMAP-compatible gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5438)
  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 5438)
  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 rtTA3-2A-Cre
FEATURES             Location/Qualifiers
     source          1..5438
                     /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"
     CDS             1091..1144
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1151..1171
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2325..2344)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2326..2344)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2351..2368)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2351..2367)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2360..2382)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2505..2807
                     /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     2686..2705
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3156..3950
                     /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             3156..3950
                     /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(3210..3229)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3820..3839
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4151..4160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4157..4531
                     /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      4669..5257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5158..5177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5411..5428
                     /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 gatcgtttac catgagtaga ctggacaagt ccaaggtcat
      361 taacggagcc ctggaactcc tgaacggggt gggcatcgag ggactgacaa caagaaagct
      421 cgcccagaag cttggagtgg agcagcctac actctactgg cacgtcaaaa acaagagagc
      481 ccttctggat gcactcccta tcgaaatgct ggacagacac cacacccatt tttgtcctct
      541 tgaaggggag tcatggcagg acttccttcg caacaacgcc aaatcatatc gctgtgcact
      601 tctgagtcat agagatggcg ccaaagtgca tctgggcacg cgccccaccg agaaacagta
      661 tgaaacactg gagaaccagc tggcctttct ctgtcaacaa ggattcagtc ttgagaatgc
      721 tctgtatgcc ctgtccgccg tggggcattt cacactggga tgcgtcctgg aagagcaaga
      781 acatcaggtt gccaaagagg aaagggagac ccccactaca gattccatgc ccccactttt
      841 gcggcaggca atcgaactgt tcgatagaca gggtgctgag cccgccttcc tgttcggatt
      901 ggaacttata atatgtggtc tggaaaaaca gctcaagtgt gaaagtggtg gtccaaccga
      961 tgctttggac gattttgacc tggacatgct gccggccgac gccctcgatg atttcgacct
     1021 tgacatgctg cctgcagatg cattggatga ctttgacctg gacatgctgc ctggtggctc
     1081 cggagccaga gagggacgag gttctttgct gacttgcggc gatgtggaag aaaacccggg
     1141 gccaggcatg cccaagaaga agcgcaaagt gtctaacctc ctgacagtgc accaaaacct
     1201 cccggcactg ccagtggatg ctacttctga cgaagtgcgc aaaaatctta tggacatgtt
     1261 ccgcgaccgg caagctttta gcgaacatac ttggaagatg ctgttgagtg tctgccgctc
     1321 ttgggccgct tggtgcaaac tcaacaaccg gaagtggttt cccgctgagc ctgaggatgt
     1381 tcgggactat ctcctgtacc ttcaagcaag aggcctcgca gtgaagacta tccaacagca
     1441 cctgggccag ctgaacatgc tgcatcggag aagcggattg ccccgaccct ccgatagcaa
     1501 tgcagttagt ttggtaatga ggagaatcag gaaagaaaat gtggatgctg gagagagggc
     1561 caaacaggct ttggcattcg aaagaaccga tttcgaccag gtccgtagcc tgatggagaa
     1621 ctcagatagg tgccaagata taagaaacct ggcctttctt ggcatcgctt ataacacact
     1681 gctgcgcatt gccgagatcg cccgcatccg ggtgaaggac atcagcagaa ctgatggcgg
     1741 caggatgctc atccatatcg gccggactaa aacactggtc tccaccgccg gggtcgagaa
     1801 ggctctgagc ctcggcgtta ccaagctcgt ggaaagatgg atcagcgtgt ctggtgtggc
     1861 cgacgaccca aacaactacc tgttttgccg agtgagaaaa aatggcgtgg cagccccttc
     1921 tgcaacttct caactgagta ctcgcgcact cgaagggatc tttgaggcta ctcaccggct
     1981 gatctacggt gctaaggacg atagcggaca gcggtatctg gcctggtccg gacacagcgc
     2041 acgagtcggc gctgcccggg acatggctag ggctggggtg tccattcccg agattatgca
     2101 agctgggggc tggactaacg ttaatatcgt gatgaactac atcagaaatc ttgactccga
     2161 aacaggcgct atggtgcggc tgttggaaga cggagactaa aaacgttgtt gtttggggtt
     2221 gaattactct ggatccacta gtaacggccg ccagtgtgct ggaattctgc agatatccat
     2281 cacactggcg gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt
     2341 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2401 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2461 gcaccgatcg cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca
     2521 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     2581 cgccggggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     2641 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     2701 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     2761 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga
     2821 gattatcaaa aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac
     2881 ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
     2941 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt
     3001 tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
     3061 cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa
     3121 gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3181 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3241 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3301 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3361 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3421 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3481 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3541 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3601 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3661 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3721 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3781 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     3841 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     3901 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg
     3961 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4021 tacaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4081 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca
     4141 cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     4201 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     4261 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     4321 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     4381 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     4441 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     4501 cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt tttaatttaa
     4561 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4621 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4681 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4741 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4801 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4861 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4921 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4981 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5041 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5101 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5161 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5221 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5281 acggttcctg ggcttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5341 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5401 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
