LOCUS       sequence-95179-e        5129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Splitposon in KanR ColE1 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5129)
  AUTHORS   Segall-Shapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA
  TITLE     A 'resource allocator' for transcription based on a highly 
            fragmented T7 RNA polymerase.
  JOURNAL   Mol Syst Biol. 2014 Jul 30;10(7):742.
  PUBMED    25080493
REFERENCE   2  (bases 1 to 5129)
  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: "Mol Syst 
            Biol."; date: "2014-07-30"; volume: "10(7)"; pages: "742"
COMMENT     Sequence Label: pTHSSd_1
FEATURES             Location/Qualifiers
     source          1..5129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(149..168)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             160..975
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQERLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(233..252)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1280..1309)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     misc_feature    1356..1377
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(1412..2494)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2456..2475
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2495..2572)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        join(3091..3096,3097..3112,3113..3119)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3125..3144
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     primer_bind     complement(3215..3233)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3395..4054
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3461..3480)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4075..4169
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4352..4940
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4841..4860
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5094..5111
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agatctgatt gattgaacga aaaacgcgaa agcgtttcac gataaatgcg aaaacggatc
       61 ctgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
      121 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
      181 acgtcttgtt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
      241 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcggttgta tgggaagccc
      301 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
      361 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
      421 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
      481 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
      541 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
      601 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
      661 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa gctgttgcca
      721 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
      781 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
      841 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca ggaacggctt
      901 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
      961 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg
     1021 acttgacggg acggcggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga
     1081 tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca
     1141 actggtccac ctacaacaac tccggcaaaa aaacgggcaa ggtgtcacca ccctgccctt
     1201 tttctttaaa accgaaaaga ttacttcgcg tttgccacct gacgtctaag aaaaggaata
     1261 ttcagcaatt tgcccgtgcc gaagaaaggc ccacccgtga aggtgagcca gtgagttgat
     1321 tgctacgtaa ttagttagtt agcccttagt gactcgaatt cgcggccgct tctagagcaa
     1381 ttcgcgctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1501 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     1561 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     1621 cctgtttgat ggtggttgac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     1681 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     1741 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     1801 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     1861 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     1921 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     1981 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     2041 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     2101 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     2161 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     2221 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     2281 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     2341 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     2401 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     2461 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     2521 ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcaacgtaaa
     2581 tgcatgccgc ttcgccttcg cgcgcgaatt ggccgccatg ccggcgataa tggcctgctt
     2641 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     2701 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     2761 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     2821 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     2881 ggctctcaag ggcatcggtc gagatccggg cttatcgact gcacggtgca ccaatgcttc
     2941 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     3001 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     3061 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     3121 gtggaattgt gagcgctcac aattaggatc cgttttcgca tttatcgtga aacgctttcg
     3181 cattttgagt gaggtatatg agatctgaaa ttctgcctcg tgatacgcct atttttatag
     3241 gttaatgtca tgataataat ggtttcttag acgtcaagtg gcactcgagt tgatcgggca
     3301 cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga
     3361 gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     3421 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     3481 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     3541 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     3601 tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     3661 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     3721 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     3781 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     3841 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     3901 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     3961 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     4021 acagtactgc gatgagtggc agggcggggc gtaatttgat atcgagctcg cttggactcc
     4081 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
     4141 gccgccgggc gttttttatt ggtgagaatc caagcctcga gctgtcagac caagtttact
     4201 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4261 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4321 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4381 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4441 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4501 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4561 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4621 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4681 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4741 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4801 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4861 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4921 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4981 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5041 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5101 cagtgagcga ggaagcctgc ataacgcga
//
