LOCUS       sequence-310860-        4849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid vector for testing DNA binding proteins by the I-Block 
            assay..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4849)
  AUTHORS   Szentes S, Zsibrita N, Koncz M, Zsigmond E, Salamon P, Pletl Z, Kiss
            A
  TITLE     I-Block: a simple Escherichia coli-based assay for studying 
            sequence-specific DNA binding of proteins.
  JOURNAL   Nucleic Acids Res. 2020 Mar 18;48(5):e28. doi: 10.1093/nar/gkaa014.
  PUBMED    31980824
REFERENCE   2  (bases 1 to 4849)
  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: "Nucleic 
            Acids Res."; date: "2020-03-18"; volume: "48(5):e28. doi"; pages: " 
            10.1093/nar/gkaa014"
COMMENT     Sequence Label: pdUA-lacIN
FEATURES             Location/Qualifiers
     source          1..4849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..78
                     /gene="lacI"
                     /label=lacI promoter
     CDS             79..1161
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(98..117)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     1210..1230
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1311..1397
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1935..2885)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2933..3155
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3647..3741)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3772..4566)
                     /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(3883..3902)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4493..4512
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4745..4763
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gctagcgccc ggaagagagt
       61 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
      121 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
      181 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
      241 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
      301 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
      361 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
      421 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
      481 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
      541 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
      601 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
      661 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
      721 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
      781 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
      841 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
      901 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
      961 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     1021 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     1081 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     1141 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg aattaaagag
     1201 agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga
     1261 actatacaaa taaatgtcca gacctgcagg catgcaagct ctagaggcat caaataaaac
     1321 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1381 tcctgagtag gacaaatccg ccgccctaga cctagggtac gggttttgct gcccgcaaac
     1441 gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc agccggtttg
     1501 ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg ggaggcgtca
     1561 ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt ttcaggctgt
     1621 ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt
     1681 gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca tctgttacat
     1741 tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa agctctgatg
     1801 tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc tttgatatgt
     1861 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
     1921 gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag tgtggttcgt
     1981 tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg catgtcactc
     2041 aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt
     2101 tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat tttgtcacca
     2161 ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct atctagttca
     2221 acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa tttcatattg
     2281 ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag ccttttaaac
     2341 tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct aatctctata
     2401 tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata aatcctcata
     2461 gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa tttttttaac
     2521 tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc gcttgagaac
     2581 ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt cctgatttcc
     2641 acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc attttcccta
     2701 ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg ttctttcctt
     2761 gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag cttggtttca
     2821 tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac aactaattca
     2881 gacatacatc tcaattggtc taggtgattt taatcactat accaattgag atgggctagt
     2941 caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt ctgctagacc
     3001 tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag acctttgtgt
     3061 gttttttttg tttatattca agtggttata atttatagaa taaagaaaga ataaaaaaag
     3121 ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt tccgcagtat
     3181 tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta aaaccctaaa
     3241 ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt tgtctccgac
     3301 catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc tcacggctct
     3361 ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact
     3421 acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg cgggtctgct
     3481 atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg
     3541 accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag
     3601 cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc tcaccaataa
     3661 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     3721 ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa
     3781 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     3841 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     3901 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     3961 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     4021 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     4081 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     4141 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     4201 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     4261 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     4321 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     4381 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     4441 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     4501 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     4561 aatcatgcga aacgatcctc atcctgtctc ttgatcagat cttgatcccc tgcgccatca
     4621 gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga
     4681 gggcgcccca gctggcaatt ccgacgtcta agaaaccatt attatcatga cattaaccta
     4741 taaaaatagg cgtatcacga ggccctttcg tcttcacctc gagatctatc gatgcatgcc
     4801 atggtaccta ttctggtggc cggaaggcga agcggcatgc atttacgtt
//
