LOCUS       sequence-198710-        5416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing Cre-2A-GFP for generation of 
            GMAP-compatible gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5416)
  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 5416)
  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 Cre-2A-GFP
FEATURES             Location/Qualifiers
     source          1..5416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        133..151
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     133..150
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             229..249
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             250..1275
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     CDS             1294..1347
                     /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             join(1354..1356,1357..1359,1360..2073)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1354..1356,1357..1359,1360..2070)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1660..1679)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2007..2028
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2198..2217)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2199..2217)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2224..2241)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2224..2240)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2233..2255)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2378..2680
                     /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     2559..2578
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3029..3823
                     /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             3029..3823
                     /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(3083..3102)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3693..3712
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4023..4032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4029..4403
                     /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      4541..5129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5030..5049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5283..5300
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac
      181 cgagctcgga tcctaactcg aacgctagct gtgcgatcgt ttaccatgcc caagaagaag
      241 aggaaggtgt ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca
      301 acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct
      361 gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg
      421 aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt
      481 caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt
      541 catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg
      601 cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa
      661 cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata
      721 cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc
      781 aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc
      841 agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact
      901 aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg
      961 ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact
     1021 cgcgccctgg aagggatttt tgaagcaact caccgattga tttacggcgc taaggatgac
     1081 tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat
     1141 atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta
     1201 aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg
     1261 ctggaagatg gcgatgggcc ggccggctcc ggcgagggca ggggaagtct tctaacatgc
     1321 ggggacgtgg aggaaaatcc cggcccagaa ttcatggtga gcaagggcga ggagctgttc
     1381 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1441 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1501 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1561 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1621 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1681 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1741 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1801 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1861 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1921 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1981 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2041 atcactctcg gcatggacga gctgtacaag taaaaacgtt gttgtttggg gttgaattac
     2101 tctggatcca ctagtaacgg ccgccagtgt gctggaattc tgcagatatc catcacactg
     2161 gcggccgctc gagcatgcat ctagagggcc caattcgccc tatagtgagt cgtattacaa
     2221 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     2281 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2341 tcgcccttcc caacagttgc gcagcctata cgtacggcag tttaaggttt acacctataa
     2401 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgccggg
     2461 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     2521 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     2581 cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     2641 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggca tgagattatc
     2701 aaaaaggatc ttcacctaga tccttttcac gtagaaagcc agtccgcaga aacggtgctg
     2761 accccggatg aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag
     2821 aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac
     2881 agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa
     2941 agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat caagctctga
     3001 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3061 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3121 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3181 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     3241 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3301 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3361 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3421 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3481 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3541 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3601 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3661 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3721 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3781 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaattatta acgcttacaa
     3841 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatcaggtg
     3901 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3961 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatag cacgtgagga
     4021 gggccaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
     4081 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
     4141 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
     4201 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt
     4261 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
     4321 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact
     4381 tcgtggccga ggagcaggac tgacacgtgc taaaacttca tttttaattt aaaaggatct
     4441 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4501 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4561 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4621 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4681 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4741 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4801 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4861 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4921 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4981 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5041 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5101 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5161 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5221 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5281 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     5341 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5401 gtgagcgcaa cgcaat
//
