LOCUS       sequence-226811-        7000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cyth2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7000)
  TITLE     Kahn expression plasmids
REFERENCE   2  (bases 1 to 7000)
  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: g5s pD42
FEATURES             Location/Qualifiers
     source          1..7000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     87..106
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        87..105
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    106..130
                     /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    171..195
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1414..1438)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1458..1499
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1509..1526
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1583..1601)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1597..1644
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1819..1824,1825..1841,1842..1847))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1878..1896)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1964..2068
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2069..2137,2138..2929)
                     /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(2287..2306)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3100..3688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3589..3608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3842..3859
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3874..4013
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3939..3958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4099..4121
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4115..4306)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5618..6700)
                     /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     6662..6681
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6701..6778)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6984..7000,1..3)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
       61 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
      121 taacaattcc cctctagaaa taattttgtt taactttaag aaggaatatc acaagtttgt
      181 acaaaaaagc aggcttagaa ggagatagaa ccatggagga cggcgtctat gaacccccag
      241 acctgactcc ggaggagcgg atggagctgg agaacatccg gcggcggaag caggagctgc
      301 tggtggagat tcagcgcctg cgggaggagc tcagtgaagc catgagcgag gtggaggggc
      361 tggaggccaa tgagggcagt aagaccttgc aacggaaccg gaagatggca atgggcagga
      421 agaagttcaa catggacccc aagaagggga tccagttctt ggtggagaat gaactgctgc
      481 agaacacacc cgaggagatc gcccgcttcc tgtacaaggg cgaggggctg aacaagacag
      541 ccatcgggga ctacctgggg gagagggaag aactgaacct ggcagtgctc catgcttttg
      601 tggatctgca tgagttcacc gacctcaatc tggtgcaggc cctcaggcag tttctatgga
      661 gctttcgcct acccggagag gcccagaaaa ttgaccggat gatggaggcc ttcgcccagc
      721 gatactgcct gtgcaaccct ggggttttcc agtccacaga cacgtgctat gtgctgtcct
      781 tcgccgtcat catgctcaac accagtctcc acaatcccaa tgtccgggac aagccgggcc
      841 tggagcgctt tgtggccatg aaccggggca tcaacgaggg cggggacctg cctgaggagc
      901 tgctcaggaa cctgtacgac agcatccgaa atgagccctt caagattcct gaggatgacg
      961 ggaatgacct gacccacacc ttcttcaacc cggaccggga gggctggctc ctgaagctgg
     1021 gagggggccg ggtgaagacg tggaagcggc gctggtttat cctcacagac aactgcctct
     1081 actactttga gtacaccacg gacaaggagc cccgaggaat catccccctg gagaatctga
     1141 gcatccgaga ggtggacgac ccccggaaac cgaactgctt tgaactttac atccccaaca
     1201 acaaggggca gctcatcaaa gcctgcaaaa ctgaggcgga cggccgagtg gtggagggaa
     1261 accacatggt gtaccggatc tcggccccca cgcaggagga gaaggacgag tggatcaagt
     1321 ccatccaggc ggctgtgagt gtggacccct tctatgagat gctggcagcg agaaagaagc
     1381 ggatttcagt caagaagaag caggagcagc ccgacccagc tttcttgtac aaagtggtga
     1441 tcaattcgaa gcttgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     1501 gtaccggtca tcatcaccat caccattgag tttgatccgg ctgctaacaa agcccgaaag
     1561 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1621 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1681 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1741 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1801 actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattaa
     1861 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     1921 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1981 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2041 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2101 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2161 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2221 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2281 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     2341 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2401 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2461 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2521 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2581 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     2641 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2701 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2761 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2821 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2881 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2941 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3001 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3061 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3121 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3181 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3241 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3301 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3361 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3421 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3481 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3541 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3601 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3661 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3721 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3781 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3841 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3901 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3961 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4021 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4081 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4141 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4201 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4261 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4321 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4381 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4441 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4501 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4561 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4621 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4681 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4741 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     4801 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     4861 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     4921 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     4981 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     5041 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     5101 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     5161 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     5221 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     5281 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5341 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5401 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5461 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5521 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5581 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5641 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5701 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5761 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5821 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5881 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5941 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6001 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6061 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6121 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6181 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6241 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6301 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6361 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6421 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6481 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6541 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6601 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6661 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6721 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6781 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6841 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6901 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6961 tggcgagccc gatcttcccc atcggtgatg tcggcgatat
//
