LOCUS       sequence-435725-        3693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 to construct history-dependent target T2PB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3693)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 3693)
  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-T6 Circuit 1 GG1-Bxb1P-bbs1-PhiC31P-GG2
FEATURES             Location/Qualifiers
     source          1..3693
                     /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"
     protein_bind    471..518
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(608..627)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(609..627)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(634..651)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(634..650)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(643..665)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1412..2206
                     /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             1412..2206
                     /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(1466..1485)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2076..2095
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2406..2415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2412..2786
                     /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      2924..3512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3413..3432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3666..3683
                     /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 ttaggtgacg cgttagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc aggggatcgg aggtctcaag gtccctacgc
      361 ccccaactga gagaactcaa aggttacccc agttggggca ctactagtat tgtcttcaag
      421 ttgctgacct tcgtggataa atcttacagt gctacaagaa gacaactccg ggtttgtacc
      481 gtacaccact gagacggcgg tggttgacca gacaaaccac gaatagtgag accacagagt
      541 gcctgaattc tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc
      601 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
      661 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      721 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata
      781 cgtacggcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
      841 tgtacagagt gatattattg acacgccggg gcgacggatg gtgatccccc tggccagcac
      901 actggccata tcggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
      961 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     1021 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gcatgagatt
     1081 atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg
     1141 ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa
     1201 gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg
     1261 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     1321 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc
     1381 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     1441 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     1501 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1561 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     1621 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     1681 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     1741 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     1801 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     1861 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     1921 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     1981 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2041 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2101 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2161 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaatta ttaacgctta
     2221 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatcag
     2281 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2341 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tagcacgtga
     2401 ggagggccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     2461 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     2521 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     2581 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     2641 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     2701 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     2761 acttcgtggc cgaggagcag gactgacacg tgctaaaact tcatttttaa tttaaaagga
     2821 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2881 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2941 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3001 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3061 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3121 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3181 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3241 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3301 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3361 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3421 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3481 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3541 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3601 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3661 agcgcagcga gtcagtgagc gaggaagcgg aag
//
