LOCUS       sequence-306912-        7061 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 7061)
  TITLE     Collier lab plasmids
REFERENCE   2  (bases 1 to 7061)
  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"
COMMENT     Sequence Label: pET-22b DT 51E/148K
FEATURES             Location/Qualifiers
     source          1..7061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1299..1490
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1484..1506)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1592..1731
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1647..1666
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1746..1763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1917..2505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1997..2016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2676..3467,3468..3536))
                     /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     3299..3318
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3537..3641)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3667..4122)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3804..3825)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4016..4035
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4159..4206)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4202..4220
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4273..4290)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5323..5895)
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWEGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVKYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     sig_peptide     complement(5929..5994)
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     RBS             6002..6024
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6039..6063
                     /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(6063..6082)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6064..6082)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6166..6185)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6391..6468
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6469..7061,1..490)
                     /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(6488..6507)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
       61 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      121 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      181 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
      241 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
      301 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
      361 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
      541 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
      601 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
      661 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
      721 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
      781 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
      841 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
      901 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
      961 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     1021 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     1081 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     1141 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     1201 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     1261 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     1321 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     1381 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     1441 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1501 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1561 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     1621 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     1681 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     1741 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1801 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1861 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1921 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1981 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2041 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2101 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2161 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2221 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2281 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2341 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2401 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2461 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2521 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2581 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2641 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2701 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2761 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2821 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2881 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2941 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     3001 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3061 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3121 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3181 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3241 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3301 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3361 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3421 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3481 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3541 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3601 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3661 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3721 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3781 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3841 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3901 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3961 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4021 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4081 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga
     4141 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
     4201 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
     4261 agccggatct cagtggtggt ggtggtggtg ctcgaggctt ttgatttcaa aaaatagcga
     4321 tagcttagaa ttaaccttgg tgtgatctac tgttttctgg tacccaagaa cgcctatgga
     4381 atccgacgaa atttcattag aatgaatttt ctccgagctg cttctgtgaa atgccacgtg
     4441 aagattcgca tgcacaccat taccaacata aacaggagat ttagggcgac aaaaagttac
     4501 atcaccgtct atagctctgc aacgcatcct tattttccga ccatttacgg aaatatgagt
     4561 cttggactta ttaacgtcca gctttccagg aatagtcggt agtaggacac ccgcgattgg
     4621 aagcggggta ttttcagcag taatttttat gtcgtgccca ctctcccctt gaaaaccagt
     4681 tcggattatc gaatcttcaa cagtgttcca actgacagca tacccgtcat gaagaaatgg
     4741 ttgcgtttta tgccccggag aatacgcggg acgattatac gaattatgaa ctacttgaaa
     4801 taaattgata atactctcta caaaattata tgcagcgaaa ccaatatcaa ctagctctcc
     4861 taccaatgga atagcttgag caaccattaa agacgataaa gctattgatt gtgccactat
     4921 ctcttctgta ttgtggtgaa cggcaccgtc tgcaatgccc attacgctac cgataccagg
     4981 aagtatcgaa agagcagcag ttgtcttttc caaattatca gctgtttcgc tatcgataac
     5041 ttgcgcaacg tttactgccc acgccgcata gttagcccca gcgaatacag gattggtccc
     5101 agtaacggtt ttaagttctg acaattcagg atgctctaat gccgtttgat gaaattcttc
     5161 taggtattgt ttagcttttt cctcagatac tgttttattg ggactttcgc tcattttatt
     5221 tttgataggg ccatgctctt tcaaagactc tatctttgtc ttagttttat cccttatgac
     5281 atcccaatca agatttatgc atgacaatga gctacctact gatcgcctga cacgatttcc
     5341 tgcacaggct tgagccatat actcatacat cgcatcttgg ccacgttttc cacgggtttc
     5401 aaaattaatc tcaagttcta cgcttaacgc tttcgcctgt tcccagttat taatatactt
     5461 aacgctagaa ctcccctcag cgaagggaag gctgagcact acacgcgaag caccatcacc
     5521 gaaccttttg ataaactctt ccgttccgac ttgctccatc aacggttcag tgagacttaa
     5581 acctaactct ttcttaatag tttcggcatt atccactttt agtgcgagaa ccttcgtcag
     5641 tcctggatac gtcactttga ccacgcctcc agcttttcca gagagcgggt tttcattatc
     5701 tacagagtat cccgcagcgt cgtatttatt gtcggtacta taaaacccct cccaatcatc
     5761 gtcataattt ccttgtgtac cagattttgg cttttgtata cctttttgaa tggaatctac
     5821 ataaccaggt ttagtcccgt ggtacgaaga aaagttttcc atcacaaaag atttagaaga
     5881 atcaacaaca tcatcagcgc ccggatccga attaattccg atatccatgg ccatcgccgg
     5941 ctgggcagcg aggagcagca gaccagcagc agcggtcggc agcaggtatt tcatatgtat
     6001 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
     6061 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     6121 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     6181 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     6241 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     6301 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     6361 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     6421 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     6481 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     6541 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     6601 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     6661 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     6721 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     6781 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     6841 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     6901 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     6961 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     7021 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc t
//
