LOCUS       sequence-226803-        7344 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 7344)
  TITLE     Kahn expression plasmids
REFERENCE   2  (bases 1 to 7344)
  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 pD47
FEATURES             Location/Qualifiers
     source          1..7344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        109..488
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        489..692
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     643..663
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     737..756
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        737..755
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    788..812
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2031..2055)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2066..2068,2069..2071,2072..2785)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2099..2127)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2722..2742
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2807..2824)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2813..3037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3083..3511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3170..3189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3380..3401
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3520..3540)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3525..3854
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3705..3840
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3767..3786
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3921..4715
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3975..3994)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4585..4604
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4891..5012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4928..4947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4982..5001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5061..5077)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5061..5077)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5074..5096)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5085..5101
                     /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)."
     promoter        complement(join(5109..5115,5116..5133,5134..5139))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5154..5175
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5292..5309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5463..6051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5543..5562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6222..7013,7014..7082))
                     /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     6845..6864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7083..7187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7262..7281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
       61 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
      121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      361 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      421 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      481 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      541 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      601 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      661 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
      721 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagttaagct
      781 tgatcaaaca agtttgtaca aaaaagcagg cttagaagga gatagaacca tggaggacgg
      841 cgtctatgaa cccccagacc tgactccgga ggagcggatg gagctggaga acatccggcg
      901 gcggaagcag gagctgctgg tggagattca gcgcctgcgg gaggagctca gtgaagccat
      961 gagcgaggtg gaggggctgg aggccaatga gggcagtaag accttgcaac ggaaccggaa
     1021 gatggcaatg ggcaggaaga agttcaacat ggaccccaag aaggggatcc agttcttggt
     1081 ggagaatgaa ctgctgcaga acacacccga ggagatcgcc cgcttcctgt acaagggcga
     1141 ggggctgaac aagacagcca tcggggacta cctgggggag agggaagaac tgaacctggc
     1201 agtgctccat gcttttgtgg atctgcatga gttcaccgac ctcaatctgg tgcaggccct
     1261 caggcagttt ctatggagct ttcgcctacc cggagaggcc cagaaaattg accggatgat
     1321 ggaggccttc gcccagcgat actgcctgtg caaccctggg gttttccagt ccacagacac
     1381 gtgctatgtg ctgtccttcg ccgtcatcat gctcaacacc agtctccaca atcccaatgt
     1441 ccgggacaag ccgggcctgg agcgctttgt ggccatgaac cggggcatca acgagggcgg
     1501 ggacctgcct gaggagctgc tcaggaacct gtacgacagc atccgaaatg agcccttcaa
     1561 gattcctgag gatgacggga atgacctgac ccacaccttc ttcaacccgg accgggaggg
     1621 ctggctcctg aagctgggag ggggccgggt gaagacgtgg aagcggcgct ggtttatcct
     1681 cacagacaac tgcctctact actttgagta caccacggac aaggagcccc gaggaatcat
     1741 ccccctggag aatctgagca tccgagaggt ggacgacccc cggaaaccga actgctttga
     1801 actttacatc cccaacaaca aggggcagct catcaaagcc tgcaaaactg aggcggacgg
     1861 ccgagtggtg gagggaaacc acatggtgta ccggatctcg gcccccacgc aggaggagaa
     1921 ggacgagtgg atcaagtcca tccaggcggc tgtgagtgtg gaccccttct atgagatgct
     1981 ggcagcgaga aagaagcgga tttcagtcaa gaagaagcag gagcagcccg acccagcttt
     2041 cttgtacaaa gtggttcgat ctagaatggc tagcaaagga gaagaacttt tcactggagt
     2101 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg
     2161 agagggtgaa ggtgatgcta catacggaaa gcttaccctt aaatttattt gcactactgg
     2221 aaaactacct gttccatggc caacacttgt cactactttc tcttatggtg ttcaatgctt
     2281 ttcccgttat ccggatcata tgaaacggca tgactttttc aagagtgcca tgcccgaagg
     2341 ttatgtacag gaacgcacta tatctttcaa agatgacggg aactacaaga cgcgtgctga
     2401 agtcaagttt gaaggtgata cccttgttaa tcgtatcgag ttaaaaggta ttgattttaa
     2461 agaagatgga aacattctcg gacacaaact cgagtacaac tataactcac acaatgtata
     2521 catcacggca gacaaacaaa agaatggaat caaagctaac ttcaaaattc gccacaacat
     2581 tgaagatgga tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     2641 ccctgtcctt ttaccagaca accattacct gtcgacacaa tctgcccttt cgaaagatcc
     2701 caacgaaaag cgtgaccaca tggtccttct tgagtttgta actgctgctg ggattacaca
     2761 tggcatggat gagctctaca aataatgaat taaacccgct gatcagcctc gactgtgcct
     2821 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2881 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2941 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3001 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3061 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3121 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3181 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3241 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3301 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3361 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3421 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3481 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3541 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3601 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3661 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3721 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3781 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3841 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3901 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3961 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4021 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4081 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4141 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4201 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4261 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4321 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4381 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4441 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4501 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4561 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4621 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4681 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg
     4741 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4801 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4861 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4921 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4981 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5041 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5101 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5161 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5641 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5881 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7201 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7261 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7321 aggtcgctga gtagtgcgcg agca
//
