LOCUS       sequence-226802-        7338 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 7338)
  TITLE     Kahn expression plasmids
REFERENCE   2  (bases 1 to 7338)
  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 pD47
FEATURES             Location/Qualifiers
     source          1..7338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        144..523
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        524..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     678..698
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     772..791
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        772..790
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    823..847
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2060..2084)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2095..2097,2098..2100,2101..2814)
                     /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(2128..2156)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2751..2771
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2836..2853)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2842..3066
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3112..3540
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3199..3218)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3409..3430
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3549..3569)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3554..3883
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3734..3869
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3796..3815
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3950..4744
                     /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(4004..4023)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4614..4633
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4920..5041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4957..4976)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5011..5030
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5090..5106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5090..5106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5103..5125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5114..5130
                     /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(5138..5144,5145..5162,5163..5168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5183..5204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5321..5338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5492..6080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5572..5591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6251..7042,7043..7111))
                     /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     6874..6893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7112..7216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7291..7310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
       61 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
      121 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
      181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      721 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
      781 tcactatagg gagacccaag ctggctagtt aagcttgatc aaacaagttt gtacaaaaaa
      841 gcaggcttag aaggagatag aaccatggag gaggacgaca gctacgttcc cagtgacctg
      901 acagcagagg agcgtcaaga actggagaac atccgacgga gaaaacagga gctgctggct
      961 gacattcaga ggctgaagga tgagatagca gaagtagcta atgaaattga aaacctggga
     1021 tccacagagg aaaggaaaaa catgcagagg aacaaacagg tagccatggg caggaaaaaa
     1081 tttaatatgg accctaaaaa ggggatccag ttcttaatag agaacgacct cctgaagaac
     1141 acttgtgaag acattgccca gttcttatat aaaggcgaag ggctcaacaa gacagccatc
     1201 ggcgactacc taggggagag agatgagttt aatatccagg ttcttcatgc atttgtggag
     1261 ctgcatgagt tcactgatct taatctcgtc caggcactac ggcagttcct gtggagcttc
     1321 cggctacccg gagaggccca gaagatcgac cggatgatgg aggcgtttgc ccagcgatat
     1381 tgtcagtgca ataatggcgt gttccagtcc acggatactt gttacgtcct ctcctttgcc
     1441 atcatcatgt tgaacaccag tctgcacaac cccaatgtca aagataagcc cactgtggag
     1501 aggttcattg ccatgaaccg aggcatcaat gatgggggag acctgccgga ggagctcctc
     1561 cggaatctct atgagagcat aaaaaatgaa ccctttaaaa tcccagaaga cgacgggaat
     1621 gacctcactc acactttctt caatccagac cgagaaggct ggctattgaa actcggaggt
     1681 ggcagggtaa agacttggaa gagacgctgg ttcattctga ctgacaactg cctttactac
     1741 tttgagtata ccacggataa ggagccccgt ggaatcatcc ctttagagaa tctgagtatc
     1801 cgggaagtgg aggactccaa aaaaccaaac tgctttgagc tttatatccc cgacaataaa
     1861 gaccaagtta tcaaggcctg caagaccgag gctgacgggc gggtggtgga ggggaaccac
     1921 actgtttacc ggatctcagc tccgacgccc gaggagaagg aggagtggat taagtgcatt
     1981 aaagcagcca tcagcaggga ccctttctac gaaatgctcg cagcacggaa aaagaaggtc
     2041 tcctccacga agcgacacga cccagctttc ttgtacaaag tggttcgatc tagaatggct
     2101 agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat
     2161 gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac atacggaaag
     2221 cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc
     2281 actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat gaaacggcat
     2341 gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat atctttcaaa
     2401 gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat
     2461 cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg acacaaactc
     2521 gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa gaatggaatc
     2581 aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact agcagaccat
     2641 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg
     2701 tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat ggtccttctt
     2761 gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa ataatgaatt
     2821 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2881 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2941 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3001 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3061 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3121 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3181 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3241 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3301 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3361 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3421 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3481 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3541 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3601 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3661 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3721 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3781 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3841 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3901 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3961 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4021 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4081 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4141 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4201 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4261 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4321 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4381 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4441 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4501 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4561 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4621 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4681 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4741 ctgagcggga ctctggggtt cgcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4801 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4861 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4921 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4981 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5041 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     7261 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7321 ccagtatctg ctccctgc
//
