LOCUS       sequence-102266-        6148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for tac promoter regulated high-level expression of cloned, 
            untagged genes in E. Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6148)
  TITLE     Arrowsmith Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6148)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pTRC-LIC
FEATURES             Location/Qualifiers
     source          1..6148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(93..161,162..953)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPTAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(311..330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1124..1712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1613..1632
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1866..1883
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1898..2038
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1964..1983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2124..2146
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2224..2301
                     /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."
     CDS             2302..3384
                     /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     complement(2321..2340)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3616..3621,3622..3638,3639..3645)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3653..3669
                     /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     3658..3680
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    complement(3720..3753)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3768..4213
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4214..4300,4301..5635)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     complement(5754..5771)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5924..6010
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6102..6129
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
       61 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      121 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      181 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      241 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      301 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      361 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      421 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      481 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      541 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      601 ccataccaaa cgacgagcgt gacaccacga tgcctacagc aatggcaaca acgttgcgca
      661 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
      721 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
      781 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
      841 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
      901 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
      961 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1021 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1081 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1141 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1201 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1261 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1321 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1381 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1441 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1501 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1561 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1621 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1681 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     1741 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     1801 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     1861 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     1921 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     1981 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2041 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2101 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2161 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcatttac
     2221 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     2281 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     2341 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     2401 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     2461 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     2521 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     2581 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     2641 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2701 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2761 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2821 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2881 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2941 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     3001 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     3061 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     3121 gacgataccg aagacagctc atgttatatc ccgccgtcaa ccaccatcaa acaggatttt
     3181 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     3241 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     3301 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3361 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc gcgaattgat
     3421 ctggtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     3481 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     3541 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     3601 ttctgaaatg agctgttgac aattaatcat ccggctcgta taatgtgtgg aattgtgagc
     3661 ggataacaat ttcacacagg aaacagacca tgattatgag ttctcctcct gaaagatcca
     3721 taacttcgta tagcatacat tatacgaagt tatgcggccg cgacgtccac atatacctgc
     3781 cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag
     3841 gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat
     3901 agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca
     3961 aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag
     4021 taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa
     4081 gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa
     4141 cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa
     4201 ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac
     4261 taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc
     4321 atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga
     4381 acagcaaaaa aatgaaaaat atcaagttcc tgagttcgat tcgtccacaa ttaaaaatat
     4441 ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac
     4501 tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc
     4561 ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg
     4621 gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct
     4681 aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg
     4741 tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca
     4801 agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc
     4861 aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa
     4921 ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca
     4981 caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc
     5041 tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa
     5101 acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat
     5161 tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac
     5221 agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt
     5281 cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat
     5341 gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct
     5401 tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt
     5461 acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta
     5521 cgcagacaaa caatcaacgt ttgcgcctag cttcctgctg aacatcaaag gcaagaaaac
     5581 atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca aataaaaacg
     5641 caaaagaaaa tgccgatatc ctattggcat tgacgtcagg tggcactttt cgaggagatc
     5701 atgcacatga tgacgaagct tggctgtttt ggcggatgag agaagatttt cagcctgata
     5761 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     5821 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt
     5881 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     5941 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     6001 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     6061 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     6121 tggccttttt gcgtttctac aaactctt
//
