LOCUS       sequence-433351-        6297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6297)
  AUTHORS   Galietta LJ, Haggie PM, Verkman AS
  TITLE     Green fluorescent protein-based halide indicators with improved 
            chloride and iodide affinities.
  JOURNAL   FEBS Lett. 2001 Jun 22. 499(3):220-4.
  PUBMED    11423120
REFERENCE   2  (bases 1 to 6297)
  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"; journalName: "FEBS Lett. 
            2001 Jun 22. 499(3):220-4."
COMMENT     Sequence Label: pcDNA3.1 Hygro EYFP H148Q/I152L
FEATURES             Location/Qualifiers
     source          1..6297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        104..483
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        484..687
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     638..658
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     732..751
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        732..750
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      817..826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(823..825,826..828,829..1542)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYLMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(868..889)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1129..1148)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1476..1497
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1588..1605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1594..1818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1864..2292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1951..1970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2161..2182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2301..2321)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2306..2635
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2486..2621
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2548..2567
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2684..3709
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2684..3709
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    3839..3960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3876..3895)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3930..3949
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4009..4025)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4009..4025)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4022..4044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4033..4049
                     /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(4057..4063,4064..4081,4082..4087))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4102..4123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4240..4257)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4411..4999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4491..4510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5170..5961,5962..6030))
                     /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     5793..5812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6031..6135)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6210..6229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
       61 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      121 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      181 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      241 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      301 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      361 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      421 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      481 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      541 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      601 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      661 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      721 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta
      781 agcttggtac cgagctcgga tccccgggta ccggtcgcca ccatggtgag caagggcgag
      841 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      901 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      961 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
     1021 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     1081 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1141 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1201 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1261 aacagccaga acgtctatct gatggccgac aagcagaaga acggcatcaa ggtgaacttc
     1321 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1381 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     1441 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1501 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg ctcgagtcta
     1561 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1621 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1681 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1741 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1801 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1861 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1921 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1981 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2041 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2101 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2161 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2221 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2281 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2341 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2401 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2461 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2521 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     2581 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2641 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatgaaaa agcctgaact
     2701 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     2761 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     2821 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     2881 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     2941 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     3001 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     3061 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     3121 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     3181 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     3241 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     3301 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     3361 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     3421 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     3481 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     3541 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     3601 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     3661 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtgctacg
     3721 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3781 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3841 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3901 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3961 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4021 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4081 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4141 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4201 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4261 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4321 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4381 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4441 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4501 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4561 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4621 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4681 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4741 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4801 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4861 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4921 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4981 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5041 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5101 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5161 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5221 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5281 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5341 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5401 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5461 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5521 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5581 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5641 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5701 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5761 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5821 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5881 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5941 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6001 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6061 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6121 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6181 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6241 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatt
//
