LOCUS       sequence-367384-        5965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HCAII in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5965)
  AUTHORS   Wang L, Hiblot J, Popp C, Xue L, Johnsson K
  TITLE     Environmentally Sensitive Color-Shifting Fluorophores for 
            Bioimaging.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Dec 1;59(49):21880-21884. doi: 
            10.1002/anie.202008357. Epub 2020 Sep 28.
  PUBMED    32762146
REFERENCE   2  (bases 1 to 5965)
  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: 
            "10.1002/anie.202008357"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2020-12-1- 1"; volume: "59"; issue: "49"; pages: 
            "21880-21884"
COMMENT     Sequence Label: pET51b_HCAII
FEATURES             Location/Qualifiers
     source          1..5965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..26)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     167..189
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(183..374)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(946..2028)
                     /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     1990..2009
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2029..2106)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2312..2331
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2419..2438
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2419..2437
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2438..2462
                     /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)."
     RBS             2477..2499
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2515..2538
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2545..2559
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             3379..3408
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3431..3449)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3445..3492
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3529..3984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3616..3635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3826..3847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4099..4890,4891..4959))
                     /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     4722..4741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4960..5052)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5133..5721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5622..5641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5875..5892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(5907..5965,1..81)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
       61 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
      121 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
      181 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
      241 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
      301 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
      361 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
      421 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
      481 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
      541 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
      601 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
      661 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
      721 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
      781 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
      841 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
      901 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
      961 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1021 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1081 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1141 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1201 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     1261 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1321 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1381 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1441 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1501 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1561 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1621 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1681 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     1741 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     1801 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     1861 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     1921 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     1981 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2041 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2101 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2161 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2221 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2281 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2341 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     2401 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     2461 cccctctaga aataattttg tttaacttta agaaggagat ataccatggc aagctggagc
     2521 cacccgcagt tcgaaaaggg tgcagatgac gacgacaagg taccgcatat gtcccatcac
     2581 tgggggtacg gcaaacacaa cggacctgag cactggcata aggacttccc cattgccaag
     2641 ggagagcgcc agtcccctgt tgacatcgac actcatacag ccaagtatga cccttccctg
     2701 aagcccctgt ctgtttccta tgatcaagca acttccctga ggatcctcaa caatggtcat
     2761 gctttcaacg tggagtttga tgactctcag gacaaagcag tgctcaaggg aggacccctg
     2821 gatggcactt acagattgat tcagtttcac tttcactggg gttcacttga tggacaaggt
     2881 tcagagcata ctgtggataa aaagaaatat gctgcagaac ttcacttggt tcactggaac
     2941 accaaatatg gggattttgg gaaagctgtg cagcaacctg atggactggc cgttctaggt
     3001 atttttttga aggttggcag cgctaaaccg ggccttcaga aagttgttga cgtgctggat
     3061 tccattaaaa caaagggcaa gagtgctgac ttcactaact tcgatcctcg tggcctcctt
     3121 cctgaatccc tggattactg gacctaccca ggctcactga ccacccctcc tcttctggaa
     3181 tgtgtgacct ggattgtgct caaggaaccc atcagcgtca gcagcgagca ggtgttgaaa
     3241 ttccgtaaac ttaacttcaa tggggagggt gaacccgaag aactgatggt ggacaactgg
     3301 cgcccagctc agccactgaa gaacaggcaa atcaaagctt ccttcaaagg agctccgggc
     3361 ttctcctcaa tttccgctca tcaccaccat catcaccatc accaccacta attaacctag
     3421 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     3481 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     3541 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3601 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3661 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3721 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3781 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3841 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3901 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3961 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     4021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4201 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     4981 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5041 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5101 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5161 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5221 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     5281 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5341 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5401 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5461 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5521 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5581 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5641 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5701 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5761 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5821 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5881 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5941 ggtatttcac accgcaatgg tgcac
//
