LOCUS       sequence-7385-ex        5740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5740)
  AUTHORS   Lee JY, Ahn HJ, Ha KS, Suh SW
  TITLE     Crystal structure of the TM1442 protein from Thermotoga maritima, a 
            homolog of the Bacillus subtilis general stress response 
            anti-anti-sigma factor RsbV.
  JOURNAL   Proteins. 2004 Jul 1. 56(1):176-9.
  PUBMED    15162498
REFERENCE   2  (bases 1 to 5740)
  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: "Proteins. 
            2004 Jul 1. 56(1):176-9."
COMMENT     Sequence Label: TM1442-pET-21a
FEATURES             Location/Qualifiers
     source          1..5740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             544..566
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    581..605
                     /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     complement(605..624)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(606..624)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(708..727)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        933..1010
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1011..2093
                     /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(1030..1049)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2902..3093
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3087..3109)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3195..3337
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3250..3269
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3352..3369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3523..4111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3603..3622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4282..5073,5074..5142))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4905..4924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5143..5247)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5274..5729)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5411..5432)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5623..5642
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcctcacgcc tcctccactg tatcggtgat tttgaagatc
      241 ttatcgagat gggtaagttt tagtattctc gatatgcttt ctttcagtga gctgaggatg
      301 aactcctttc cgttgatttt cgcgtccttc aaaatcacaa cgagtgttcc aaggcccgcg
      361 ctgtccatgt aggagacaga cgaaagatcg aggacgatct tcttcttgct ggtggtcgag
      421 atgaagtttc taagctgttc cttcaactcc gaagaattgt aagcgtctat gtccccctgc
      481 actctcacga tggctttgtc atcctgttca acgatatcga gtttgaggtt gttcatatgt
      541 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
      601 attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg
      661 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
      721 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
      781 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
      841 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
      901 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
      961 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     1021 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     1081 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     1141 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     1201 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     1261 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     1321 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     1381 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     1441 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     1501 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     1561 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     1621 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     1681 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     1741 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     1801 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     1861 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     1921 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     1981 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2041 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2101 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     2161 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2221 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2281 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2341 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2401 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     2461 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2521 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     2581 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     2641 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     2701 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     2761 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     2821 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     2881 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     2941 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3001 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     3061 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     3121 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     3181 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     3241 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat
     3301 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     3361 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3421 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3481 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3541 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3601 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3661 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3721 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3781 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3841 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3901 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3961 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4021 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4081 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4141 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4201 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4261 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4321 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4381 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4441 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4501 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4561 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     4621 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4681 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4741 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4801 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4861 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4921 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4981 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5041 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5101 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5161 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5221 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaaattgt
     5281 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5341 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5401 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5461 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5521 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5581 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5641 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5701 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
