LOCUS       sequence-162300-        6375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  an updated version of cLac143 
            (pACSA_PcptOO_C38T,A39T_YFP_PMB2_LacIWF). The homologous region 
            upstream of the inducible system was fixed to add a stop codon for 
            A1837. No difference in expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6375)
  AUTHORS   Markley AL, Begemann MB, Clarke RE, Gordon GC, Pfleger BF
  TITLE     Synthetic biology toolbox for controlling gene expression in the 
            cyanobacterium Synechococcus sp. strain PCC 7002.
  JOURNAL   ACS Synth Biol. 2015 May 15;4(5):595-603. doi: 10.1021/sb500260k. 
            Epub 2014 Sep 25.
  PUBMED    25216157
REFERENCE   2  (bases 1 to 6375)
  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"; doi: "10.1021/sb500260k";
            journalName: "ACS Synth Biol"; date: "2015-05-15- 15"; volume: "4"; 
            issue: "5"; pages: "595-603"
COMMENT     Sequence Label: pALM179
FEATURES             Location/Qualifiers
     source          1..6375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(728..1810)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1772..1791
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1840..1874)
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     terminator      complement(1972..2019)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2015..2033
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(2103..2816,2817..2819,2820..2822))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2148..2169)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2497..2516
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2756..2777
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2844..2855)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2868..2897)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     protein_bind    2946..2962
                     /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    3047..3066
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     primer_bind     complement(3778..3795)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3949..4537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4029..4048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4708..5499,5500..5568))
                     /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     5331..5350
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5569..5673)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5751..6206)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5888..5909)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6100..6119
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6332..6354
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6346..6363
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6347..6363
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6370..6375,1..14)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6370..6375,1..13)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gactcactat agggcgaatt ctggcaaaca actttggctg ccgtcccacc attgagtgcg
       61 gcagtcacct ggcaaacaac tttggctgcc gtcccaccat cgctggcaaa caactttggc
      121 tgccgtccca ccatcgctgg caaacaactt tggctgccgt cccaccatcg ccaacccaag
      181 caacggcgat cgccacggtg caagacaatt gggacatggc cctagcccaa cagcccaacg
      241 accccgaaat tttgatggcc ctgggttttg tccattacct caatgcagat tacgaagccg
      301 ccaatgatcg ttggtatgaa gccttagcgg cggcaacgac ccaggatttc cccccagccc
      361 tccaggcgga gatttatgcg ggcctggcga ttagtatgta cgatttggcc caaaggcaac
      421 ccaccgacgt tgaccaggaa attcttgtag ggaaagccga aaaactccgg gcgatcgccc
      481 tagatctcga tcgttcccag ctcaaccccc gccgcctcga acaaagttgg ttatggttcc
      541 ctgaggcgat cgccacttgg caaaccctgc tcgctttaga agaatctccc taggtcatat
      601 ccggggcata cattcgatca acttgagcgc gaaccggaag gagctgactg ggttgaaggc
      661 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
      721 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
      781 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
      841 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
      901 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      961 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1021 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1081 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1141 acatggcact aaagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1201 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1261 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1321 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1381 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1441 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1501 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     1561 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     1621 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     1681 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     1741 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     1801 ctggtttcac attcaccacc atgaattgac cctcctccgg ctagcattgt acctaggact
     1861 gagctagctg taaatgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1921 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgatctag acaaaaaacc
     1981 cgtcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag cggtggcagc
     2041 agccaactca gcttcctttc gggctttgtt agcagccgga tcctgccaaa gcaccgccct
     2101 cgttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     2161 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg
     2221 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     2281 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     2341 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     2401 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     2461 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     2521 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     2581 tgcttcatgt ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg
     2641 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     2701 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     2761 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     2821 atcgaggctg ctgcctgcat atgacgacct tcgatatggc cgctgctgtg atgatgatga
     2881 tgatgatgat gatgatggcc catggaatta atctcctact tgactttatg agttggtgtg
     2941 tgaaattgtt atccgctcac aattccacac atttatacaa cggtgatggg acttatgtca
     3001 attacatctt gttaatttta ttcctgcttt tttgttaaga attccgaatt gtgagcgctc
     3061 acaattcgtt tggagatgtc cagaggggag tcagtaaatt ccaaagacaa aactgattcc
     3121 cccttttaaa cacaggccta ggtttgactt tagttcgttt caatgaaggc gaaacgcccc
     3181 tgttgacccg attcatccat ttcaatttgg gccacaaaaa attccttctg gacaatttcc
     3241 ccttcctctg tgaaggaaat ttcacccaaa ggcgtgacgt aggtacctgc aaaaatttcg
     3301 tcccgcagtt gtcgtcgcaa gtccggtaga gcaagagttt ctaagggcgt tttttcatcg
     3361 aggctgctga gggcttcaac aaaaacttgg atcgccgtaa aagcctgggc actaaattgg
     3421 ggcggctctt tttggttttg ttgaaaatag gcgtcccgaa acgcgcggtt aatctcatta
     3481 tctaactcgg cactgtaggc ttgggccacc aacaccccat cacattttgc ttggcagacg
     3541 gggaaaatat tagaagtatt taagccgttc cccccaacaa taatgccgtt gtaacctaat
     3601 tctcgcagtt gcttcaccag gttgcccgca tcggctgcta accccgaaat aatcactaga
     3661 tctggttgaa cattgagtgc ggcagttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3721 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3781 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3841 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3901 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3961 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4021 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4081 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4141 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4201 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4261 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4321 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4381 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4441 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4501 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4561 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4621 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4681 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4741 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4801 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4861 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4921 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4981 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5041 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5101 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5161 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5221 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5281 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5341 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5401 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5461 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5521 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5581 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5641 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5701 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag
     5761 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5821 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5881 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5941 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     6001 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6061 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6121 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6181 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa
     6241 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6301 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     6361 agtgaattgt aatac
//
