LOCUS       sequence-434130-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28b derived T7 promoter with a 5'-UTR transcriptional fusion to 
            clac143.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  AUTHORS   Jones CM, Korosh TC, Nielsen DR, Pfleger BF
  TITLE     Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 
            7002 mirrors the protein overproduction phenotype from E. coli 
            BL21(DE3).
  JOURNAL   Appl Microbiol Biotechnol. 2021 Feb;105(3):1147-1158. doi: 
            10.1007/s00253-020-11085-x. Epub 2021 Jan 14.
  PUBMED    33443634
REFERENCE   2  (bases 1 to 7354)
  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"; doi: 
            "10.1007/s00253-020-11085-x"; journalName: "Appl Microbiol 
            Biotechnol"; date: "2021-02"; volume: "105"; issue: "3"; pages: 
            "1147-1158"
COMMENT     Sequence Label: pCMJ074
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(175..243,244..1035)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(393..412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1206..1794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1695..1714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1948..1965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2082..2103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2118..2123,2124..2141,2142..2148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2156..2172
                     /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     2180..2196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2180..2196
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2831..2850
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2934..2953
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2934..2952
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2957..2973
                     /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)."
     CDS             3022..3051
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3064..3075
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(3097..3099,3100..3102,3103..3816)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3142..3163)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3403..3422)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3750..3771
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3866..3884)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3880..3927
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4062..4096
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     CDS             4126..5208
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4145..4164)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             complement(5489..6304)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5489..6304)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6212..6231
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6296..6315
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(6934..6951)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6934..6950)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6943..6965)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7159..7178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7278..7300
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(join(7338..7354,1..2))
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
       61 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      121 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      181 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      241 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      301 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      361 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      421 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      481 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
      541 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
      601 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
      661 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
      721 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
      781 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
      841 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
      901 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
      961 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1021 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1201 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1261 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1321 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1381 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1441 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1501 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1561 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1621 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1681 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1741 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1801 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     1861 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     1921 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     1981 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2041 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     2101 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2161 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc
     2221 ctgcaggtcg actaaaggca atctcgttat caaaagaagg cagttggtct tggagcttga
     2281 ttaattcttc gagaaagtcg ggacgaatca agtctggccg ggttgaaagg gcttgcccta
     2341 ctttgataaa tgtcggccct aagcgggtca gaatttcacg cagttcgttg gcgcgacggg
     2401 cgcgggtttc gggttcttct tcgcgtttac tatctagcca caatttgaag gaaaaccacc
     2461 caaataacca aatgatcgta aaggcccgcc acagggattg ccagggtcgc caacggtaat
     2521 gtttggcgat cgccttggcg tcatagcggg tcaaggtggt agaaccaagt ttgtggtcgg
     2581 gggcaacgga aggatactgg gtcacagtgg tttcgccgtc aatgaattgt gtggttttgt
     2641 aaagtaaatc acactatatt gtatcgaaat ttttcgcagc gacaccaaac caagcctgca
     2701 tcggggatca agcacagcca ttttctcgcc caatggcgcc caacagtccc ccggccacgg
     2761 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     2821 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     2881 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     2941 actcactata ggggaattgt gagcggataa caatttcaca caccaactca taaagtcaag
     3001 taggagatta attccatggg ccatcatcat catcatcatc atcatcatca cagcagcggc
     3061 catatcgaag gtcgtcatat gcaggcagca gcctcgatgg tgagcaaggg cgaggagctg
     3121 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3181 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3241 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
     3301 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3361 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3421 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3481 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3541 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3601 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3661 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     3721 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3781 gggatcactc tcggcatgga cgagctgtac aagtgagatc cggctgctaa caaagcccga
     3841 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3901 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     3961 ggggtctaga tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     4021 aagggagagc gtcgagatcc cggacaccat cgaatggcgc atttacagct agctcagtcc
     4081 taggtacaat gctagccgga ggagggtcaa ttcatggtgg tgaatgtgaa accagtaacg
     4141 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4201 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4261 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4321 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4381 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4441 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4501 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     4561 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     4621 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     4681 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     4741 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actttagtgc catgtccggt
     4801 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     4861 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     4921 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     4981 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5041 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5101 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5161 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5221 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     5281 caacccagtc agctccttcc ggttcgattt tgccgatttc ggcctattgg ttaaaaaatg
     5341 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     5401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5461 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     5521 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     5581 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     5641 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     5701 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     5761 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     5821 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     5881 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     5941 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     6001 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     6061 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     6121 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     6181 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     6241 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     6301 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     6361 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtaggtta agcgaaactt
     6421 tataatgttc agagagtaaa taaacctagc gtcaaaccat ctaaaagcca atcactgcaa
     6481 ctcataagca acggtgattc aggagattgg aaaaacgcaa tgcacaaaca aaccatctca
     6541 ccccatcacc agatttaatt taaataagca taacggaaga cctgcggatc catgacaaga
     6601 attgaaactc gtaccgaacc tatggtgatt aacatggggc ctcaccaccc ctccatgcac
     6661 ggcgtattac gcttgatcgt cacccttgac ggggaagacg tcgttgattg cgaacccgtc
     6721 atcggctatc tgcatcgggg tatggaaaaa attgccgaaa atcgcaccaa tgtcatgtat
     6781 gtcccctatg tgagtcgttg ggactacgcc gcagggatgt ttaacgaagc gatcaccgtt
     6841 aatgcccccg aaaaactcgc cgatatcgaa gtgccgaagc gcgcccaata tatccgcgtg
     6901 atcataggat ccccgggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga
     6961 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     7021 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     7081 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     7141 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     7201 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     7261 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     7321 acgcgcgaga cgaaagggcc tcgtgatacg ccta
//
