LOCUS       sequence-437905-        6277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of Gal4-DBD HOXD4 AroPERFECT shuffled 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6277)
  AUTHORS   Naderi J, Magalhaes AP, Kibar G, Stik G, Zhang Y, Mackowiak SD, 
            Wieler HM, Rossi F, Buschow R, Christou-Kent M, Alcoverro-Bertran M,
            Graf T, Vingron M, Hnisz D
  TITLE     An activity-specificity trade-off encoded in human transcription 
            factors.
  JOURNAL   Nat Cell Biol. 2024 Aug;26(8):1309-1321. doi: 
            10.1038/s41556-024-01411-0. Epub 2024 Jul 5.
  PUBMED    38969762
REFERENCE   2  (bases 1 to 6277)
  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"; doi: 
            "10.1038/s41556-024-01411-0"; journalName: "Nat Cell Biol"; date: 
            "2024-08"; volume: "26"; issue: "8"; pages: "1309-1321"
COMMENT     Sequence Label: pGAL4-DBD-HOXD4-AroPERFECT_shuffled2
FEATURES             Location/Qualifiers
     source          1..6277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(92..421)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      106..241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(160..179)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     406..426
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(435..863)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(545..566)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     757..776
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    909..1133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1122..1139
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        1159..1177
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1159..1176
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1623..2063)
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     primer_bind     complement(2125..2144)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2126..2144)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2189..2392)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2218..2238)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2393..2772
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2944..2963
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3038..3142
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3143..3211,3212..4003)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3361..3380)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4174..4762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4663..4682
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4916..4933
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5050..5071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5086..5091,5092..5109,5110..5116)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5124..5140
                     /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     5129..5151
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5148..5164
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5148..5164
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5213..5334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5224..5243)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5278..5297
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5508..6277,1..25))
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5619..5638)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6229..6248
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
       61 cgaaaatgga tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc
      121 ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa
      181 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
      241 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
      301 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
      361 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
      421 cattccacag aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
      481 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
      541 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
      601 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
      661 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
      721 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
      781 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
      841 taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc
      901 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
      961 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     1021 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     1081 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     1141 tgatcagcga gctctagcat ttaggtgaca ctatagaata gggccctcta gacctaacct
     1201 tttcctggct gccattggct gctagggcct gggtggctat acctacctgg tggtggctgg
     1261 gagtggtagg ccaactttcc cgcgctggct tggtactgct ccccacaggg cttgtcacca
     1321 tacagtgctc caccaccctg cttatcgtat tcttccaatc ctcccatagg gggatatggt
     1381 gactggccag ggctgccctc gtaagcatct ggcgcagcca ccccttcgaa catcggggat
     1441 gggggccctg gtacgaacac accagctgcc gttggaccag cgaataaggg caccagaccg
     1501 tcgtcggggt aaggacccgg catctgcggg ccgagataag ggttagggca cgcttcccta
     1561 gcaaatcgca ccacgccctg ctttggttta tatggagccc caggcgcagg ccccatgatc
     1621 gccgatacag tcaactgtct ttgacctttg ttactactct cttccgatga tgatgtcgca
     1681 cttattctat gctgtctcaa tgttagaggc atatcagtct ccactgaagc caatctatct
     1741 gtgacggcat ctttattcac attatcttgt acaaataatc ctgttaacaa tgcttttata
     1801 tcctgtaaag aatccatttt caaaatcatg tcaaggtctt ctcgaggaaa aatcagtaga
     1861 aatagctgtt ccagtctttc tagccttgat tccacttctg tcagatgtgc cctagtcagc
     1921 ggagaccttt tggttttggg agagtagcga cactcccagt tgttcttcag acacttggcg
     1981 cacttcggtt tttctttgga gcacttgagc tttttaagtc ggcaaatatc gcatgcttgt
     2041 tcgatagaag acagtagctt catgaattcc agcacactgg cggccgttac tagtggatcc
     2101 gagctcggta ccaagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt
     2161 aagcagtggg ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac
     2221 gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt
     2281 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     2341 gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa
     2401 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     2461 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     2521 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     2581 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     2641 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     2701 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     2761 ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa
     2821 ccctaagcag attcttcatg caattgtcgg tcaagccttg ccttgttgta gcttaaattt
     2881 tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact
     2941 atgcggcatc agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga
     3001 tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3061 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3121 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3181 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3241 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3301 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3361 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3421 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3481 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3541 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3601 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3661 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3721 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3781 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3841 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3901 ctcccgtatc gtagttatct acacgacggg aagtcaggca actatggatg aacgaaatag
     3961 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4021 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4081 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4141 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4201 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4261 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4321 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4381 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4441 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4501 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4561 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4621 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4681 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4741 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4801 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4861 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4921 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4981 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5041 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     5101 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     5161 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac
     5221 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     5281 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     5341 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     5401 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     5461 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     5521 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     5581 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     5641 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     5701 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     5761 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     5821 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     5881 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     5941 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     6001 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     6061 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     6121 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     6181 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     6241 ccgaatagcc tctccaccca agcggccgga gaacctg
//
