LOCUS       sequence-435859-        4834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 to construct history-dependent target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4834)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 4834)
  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"
COMMENT     Sequence Label: L0-T14 GG0-tUBQ1-NLS-NeonGreen-GG1
FEATURES             Location/Qualifiers
     source          1..4834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      146..155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             152..526
                     /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      664..1252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1153..1172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1406..1423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1540..1561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1576..1581,1582..1599,1600..1606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1614..1630
                     /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     1619..1641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1638..1654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1638..1654
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(2296..2316)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2353..3060)
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2796..2805
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3155..3174)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3156..3174)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3181..3198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3181..3197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3190..3212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3335..3637
                     /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     3516..3535
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3986..4780
                     /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             3986..4780
                     /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(4040..4059)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4650..4669
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
       61 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      121 gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt
      181 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
      241 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
      301 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
      361 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
      421 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
      481 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact
      541 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
      601 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      661 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      721 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      781 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
      841 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      901 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      961 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1021 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1081 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1141 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1201 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1261 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1321 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     1381 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     1441 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     1501 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     1561 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     1621 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tatttaggtg
     1681 acgcgttaga atactcaagc tatgcatcaa gcttggtacc gagctcggat ccactagtaa
     1741 cggccgccag tgtgctggaa ttcaggcact ctgtggtctc aggagcattt gtttgtcttt
     1801 aacaacggct cacaacctgt gaccgtagag agcctcaatg catggaacat gcagaagcct
     1861 ctgaagatga accaaggtgc aaagtgatca catcattctt taattttttc gttgacaaat
     1921 ttaattcatt cgttaaaaca gagagtctgt cacaatatat tgtagaaaaa aggaacctat
     1981 tgtgtttctt ttacaatcaa tacaatttgt acatagttta tcactactaa caatgtattg
     2041 atcttatatt ttattaatga aacacgtctc ctaatcacat aaacggtcat tatttcacga
     2101 tacttgtata gattggaatt aagcaagaat cttccgtaat cgattcagaa taatagaaaa
     2161 ccaaatcctt ttactgacat tatcttttaa catagaaagt cactgaaaca agaaaaacaa
     2221 accctatcaa agggaagaca acaaaaaaaa gcaagcaaga gatgggattc ttgataagag
     2281 tctctaagct tattagactt tcctcttctt ctttggagag ccagatccag atcctgatcc
     2341 tgagccactg cccttgtaca gctcgtccat gcccatcaca tcggtaaagg ccttttgcca
     2401 ctccttgaag ttgagctcgg tcttggagtg cttgagctcc gtcttacgga acacgtacat
     2461 cggctggttc ttcagatagt tagccgccat tggcttggca aaggtgtagg tggtccgcgc
     2521 agtgctccgg tagcgcttgc catttccagt ggtgtaactc cacttaaagg tactgatgat
     2581 ggttttgtcg ttggggtaag tcttcttcga cctgcaccag tccgcagcgg tcagcgagtt
     2641 ggtcatcaca ggaccgtcag cagggaaacc agtccccttc acctgggcct ctcctttgat
     2701 gtggcttccc tcgtaggtgt agcggtagtt aacagtaagg gaggcaccat cttcaaactg
     2761 cattgtgcga tggacttggt agccggagcc atctaccatg gcggcctgga aaggcgacat
     2821 cccgtcaggg tagggcaggt actgatggaa gccatacccg atatgaggga ccagaatcca
     2881 gggggagaac tggaggtcac ccttggtgga cttcaggttt aactcctcat aaccatcatt
     2941 tggattgccg gtgccctgac ccaccatgtc aaagtccaca ccgttgatgg agccaaagat
     3001 gtgtaactca tgtgtcgctg ggagagaggc catgttatcc tcctcgccct tgctcaccat
     3061 cattgtttac tattgagacc atcaacgtcc tgaattctgc agatatccat cacactggcg
     3121 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc
     3181 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3241 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3301 cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag
     3361 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg
     3421 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     3481 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     3541 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     3601 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa
     3661 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc
     3721 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
     3781 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
     3841 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
     3901 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca
     3961 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4021 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4081 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4141 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4201 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4261 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4321 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4381 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4441 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4501 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4561 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4621 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4681 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4741 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt
     4801 cctgatgcgg tattttctcc ttacgcatct gtgc
//
