LOCUS       sequence-435728-        4928 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 4928)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 4928)
  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-T13 GG3-tRBCS-mCherry-NLS-GG4
FEATURES             Location/Qualifiers
     source          1..4928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(55..849)
                     /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             complement(55..849)
                     /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(166..185)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     776..795
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1198..1500)
                     /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     complement(1300..1319)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     1623..1645
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1637..1654
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1638..1654
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1661..1680
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1661..1679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1762..2466
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(1907..1925)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2175..2194
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2476..2496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      2499..3140
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     primer_bind     complement(3275..3291)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3275..3291)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3288..3310)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3299..3315
                     /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)."
     promoter        complement(join(3323..3329,3330..3347,3348..3353))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3368..3389
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3506..3523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3677..4265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3757..3776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4403..4777)
                     /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"
     regulatory      4774..4783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa taattcagaa
       61 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
      121 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
      181 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
      241 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
      301 atcctcgccg tcgggcatgc tcgccttgag cctggcgaac agttcggctg gcgcgagccc
      361 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
      421 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
      481 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
      541 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
      601 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
      661 ctcgtcttgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
      721 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
      781 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
      841 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gagcttgatc ccctgcgcca
      901 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
      961 agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag
     1021 ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct
     1081 tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc
     1141 tttctacgtg aaaaggatct aggtgaagat cctttttgat aatctcatgc ctgacattta
     1201 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
     1261 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
     1321 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
     1381 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccc ggcgtgtcaa taatatcact
     1441 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
     1501 ccgtacgtat aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     1561 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     1621 ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg
     1681 cgaattgggc cctctagatg catgctcgag cggccgccag tgtgctggaa ttcaggcgtg
     1741 atgaggtctc aaagctaaac aatggtgagc aagggcgagg aggataacat ggccatcatc
     1801 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1861 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1921 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     1981 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     2041 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     2101 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     2161 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     2221 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     2281 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     2341 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     2401 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     2461 ctgtacagat cttaaccaaa gaagaagagg aaagtcaaag ctttcgttcg tatcatcggt
     2521 ttcgacaacg ttcgtcaagt tcaatgcatc agtttcattg cgcacacacc agaatcctac
     2581 tgagttcgag tattatggca ttgggaaaca tgtttttctt gtaccatttg ttgtgcttgt
     2641 aatttactgt gttttttatt cggttttcgc tatcgaactg tgaaatggaa atggatggag
     2701 aagagttaat gaatgatatg gtccttttgt tcattctcaa attaatatta tttgtttttt
     2761 ctcttatttg ttgtgtgttg aatttgaaaa tataagagat atgcaaacat tttgttttga
     2821 gtaaaaatgt gtcaaatcgt ggcctctaat gaccgaagtt aatatgagga gtaaaacact
     2881 tgtagttgta ccattatgct tattcactag gcaacaaata tattttcaga cctagaaaag
     2941 ctgcaaatgt tactgaatac aagtatgtcc tcttgtgttt tagacattta tgaactttcc
     3001 tttatgtaat tttccagaat ccttgtcaga ttctaatcat tgctttataa ttatagttat
     3061 actcatggat ttgtagttga gtatgaaaat attttttaat gcattttatg acttgccaat
     3121 tgattgacaa catgcatcaa tcgctgctga gaccacagag tgcctgaatt ccagcacact
     3181 ggcggccgtt actagtggat ccgagctcgg taccaagctt gatgcatagc ttgagtattc
     3241 taacgcgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3301 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3361 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3421 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3481 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3541 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3601 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3661 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3721 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3781 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3841 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3901 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3961 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4021 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4081 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4141 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4201 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4261 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4321 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4381 aaaaatgaag ttttagcacg tgtcagtcct gctcctcggc cacgaagtgc acgcagttgc
     4441 cggccgggtc gcgcagggcg aactcccgcc cccacggctg ctcgccgatc tcggtcatgg
     4501 ccggcccgga ggcgtcccgg aagttcgtgg acacgacctc cgaccactcg gcgtacagct
     4561 cgtccaggcc gcgcacccac acccaggcca gggtgttgtc cggcaccacc tggtcctgga
     4621 ccgcgctgat gaacagggtc acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga
     4681 agtcccggga gaacccgagc cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg
     4741 cggtgagcac cggaacggca ctggtcaact tggccatggt ggccctcctc acgtgctatt
     4801 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4861 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt
     4921 gaaatacc
//
