LOCUS       sequence-226676-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cyth1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  TITLE     Kahn expression plasmids
REFERENCE   2  (bases 1 to 6994)
  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: g4s pD42
FEATURES             Location/Qualifiers
     source          1..6994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        89..107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    108..132
                     /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)."
     protein_bind    173..197
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1410..1434)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1454..1495
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1505..1522
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1579..1597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1593..1640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1815..1820,1821..1837,1838..1843))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1874..1892)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1960..2064
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2065..2133,2134..2925)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2283..2302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3096..3684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3585..3604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3838..3855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3870..4009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3935..3954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4095..4117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4111..4302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5614..6696)
                     /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     6658..6677
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6697..6774)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6980..6994,1..5)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
       61 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      121 gataacaatt cccctctaga aataattttg tttaacttta agaaggaata tcacaagttt
      181 gtacaaaaaa gcaggcttag aaggagatag aaccatggag gaggacgaca gctacgttcc
      241 cagtgacctg acagcagagg agcgtcaaga actggagaac atccgacgga gaaaacagga
      301 gctgctggct gacattcaga ggctgaagga tgagatagca gaagtagcta atgaaattga
      361 aaacctggga tccacagagg aaaggaaaaa catgcagagg aacaaacagg tagccatggg
      421 caggaaaaaa tttaatatgg accctaaaaa ggggatccag ttcttaatag agaacgacct
      481 cctgaagaac acttgtgaag acattgccca gttcttatat aaaggcgaag ggctcaacaa
      541 gacagccatc ggcgactacc taggggagag agatgagttt aatatccagg ttcttcatgc
      601 atttgtggag ctgcatgagt tcactgatct taatctcgtc caggcactac ggcagttcct
      661 gtggagcttc cggctacccg gagaggccca gaagatcgac cggatgatgg aggcgtttgc
      721 ccagcgatat tgtcagtgca ataatggcgt gttccagtcc acggatactt gttacgtcct
      781 ctcctttgcc atcatcatgt tgaacaccag tctgcacaac cccaatgtca aagataagcc
      841 cactgtggag aggttcattg ccatgaaccg aggcatcaat gatgggggag acctgccgga
      901 ggagctcctc cggaatctct atgagagcat aaaaaatgaa ccctttaaaa tcccagaaga
      961 cgacgggaat gacctcactc acactttctt caatccagac cgagaaggct ggctattgaa
     1021 actcggaggt ggcagggtaa agacttggaa gagacgctgg ttcattctga ctgacaactg
     1081 cctttactac tttgagtata ccacggataa ggagccccgt ggaatcatcc ctttagagaa
     1141 tctgagtatc cgggaagtgg aggactccaa aaaaccaaac tgctttgagc tttatatccc
     1201 cgacaataaa gaccaagtta tcaaggcctg caagaccgag gctgacgggc gggtggtgga
     1261 ggggaaccac actgtttacc ggatctcagc tccgacgccc gaggagaagg aggagtggat
     1321 taagtgcatt aaagcagcca tcagcaggga ccctttctac gaaatgctcg cagcacggaa
     1381 aaagaaggtc tcctccacga agcgacacga cccagctttc ttgtacaaag tggtgatcaa
     1441 ttcgaagctt gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac
     1501 cggtcatcat caccatcacc attgagtttg atccggctgc taacaaagcc cgaaaggaag
     1561 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1621 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     1681 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     1741 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     1801 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattaattct
     1861 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1921 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1981 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2041 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2101 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2161 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2221 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2281 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2341 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2401 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2461 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2521 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2581 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2641 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2701 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2761 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2821 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2881 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2941 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3001 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3061 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3121 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3181 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3241 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3301 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3361 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3421 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3481 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3541 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3601 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3661 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3721 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3781 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3841 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3901 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3961 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4021 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4081 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4141 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4201 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4261 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4321 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4381 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4441 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4501 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4561 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4621 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4681 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4741 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg
     4801 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     4861 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     4921 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     4981 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     5041 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
     5101 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     5161 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     5221 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     5281 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5341 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5401 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5461 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5521 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5581 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5761 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5821 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5881 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5941 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6001 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6061 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6121 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6181 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6241 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6301 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6361 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6421 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6481 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6541 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6601 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6661 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6721 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6781 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6841 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6901 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6961 gagcccgatc ttccccatcg gtgatgtcgg cgat
//
